Glycan-Interacting Compounds and Methods of Use

ABSTRACT

The present invention provides glycan-interacting antibodies and methods for producing glycan-interacting antibodies useful in the treatment and prevention of human disease, including cancer. Such glycan-interacting antibodies include humanized antibodies, derivatives and fragments thereof as well as related compositions and kits. Methods of using glycan-interacting antibodies for treatment and diagnosis are included.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. Application No.17/846,389, which is a divisional application of U.S. Application No.17/539,389, filed Dec. 1, 2021, which is a divisional of U.S.Application No. 17/234,075, filed Apr. 19, 2021, which is a divisionalof U.S. Application No. 15/775,410 filed May 11, 2018, now U.S. Pat. No.11,028,181, which is a national stage entry pursuant to 35 U.S.C. §371of International Application Number PCT/US2016/061427 filed Nov. 10,2016, which claims priority to U.S. Provisional Application Number62/254,278 filed Nov. 12, 2015, U.S. Provisional Application Number62/274,572 filed Jan. 4, 2016, U.S. Application No. 62/287,666 filedJan. 27, 2016, U.S. Provisional Application No. 62/293,989 filed Feb.11, 2016, U.S. Provisional Application No. 62/345,515 filed Jun. 3,2016, and U.S. Provisional Application No. 62/382,835 filed Sep. 2,2016, each of which is incorporated by reference herein in its entiretyfor any purpose.

SEQUENCE LISTING

This application contains a Sequence Listing, which has been submittedelectronically in XML format and is hereby incorporated by reference inits entirety. Said XML copy, created on Apr. 13, 2023, is named2023-04-13_01218-0015-04US_ST26.xml and is 284,943 bytes in size.

GOVERNMENT SUPPORT

This invention was made with government support under Grant Nos.HHSN261201400027C and HHSN261201600042C awarded by the Department ofHealth and Human Services. The United States government may have certainrights in the invention.

FIELD OF THE INVENTION

This disclosure relates to glycan-interacting compounds, such asantibodies, and methods for the development of such compounds andrelated compositions for the detection and/or removal of glycosylatedmatter from an organism. The invention also relates to methods oftreating diseases related to abberant glycosylation, such as cancer,with glycan-interacting compounds and compositions presented herein.

BACKGROUND OF THE INVENTION

Aberrant glycosylation accompanies some of the other mutations commonlyobserved in carcinomas. It has been estimated that about 80% of allcarcinomas express the truncated glycans, the Tn Antigen and thesialylated form, Sialyl Tn (STn). With few exceptions, Tn and STn arenot expressed in normal, healthy tissues. Furthermore, the non-humanimmunogenic sialic acid, N-glycolylneuraminic acid (Neu5Gc), seems to bedifferentially expressed on carcinomas such as breast cancer in the formof Neu5Gc-STn (GcSTn).

Multiple aberrant glycosylation forms have been described in humancancers, identifying specific glycans as a class of cell surfacemolecules suitable for specific tumor targeting (Cheever, M.A. et al.,Clin Cancer Res. 2009 Sep 1;15(17):5323-37). For example, various humancancer types (such as bladder, breast, cervical, colon, lung, andovarian cancer among others) show high expression of STn antigen, whichis rare in normal human tissues (Karlen, P. et al., Gastroenterology.1998 Dec;11 5(6):1395-404; Ohno, S. et al, Anticancer Res. 2006Nov-Dec;26(6A):4047-53). In addition, the presence of STn ontumor-associated mucins relates to cancer with poor prognosis and istherewith considered an attractive epitope for cancer detection andtargeted therapy (Cao, Y. et al., Virchows Arch. 1997 Sep;431(3):159-66;Julien, S. et al., Br J Cancer. 2009 Jun 2;100(11):1746-54; Itzkowitz,S.H. et al., Cancer. 1990 Nov 1;66(9):1960-6; Motoo, Y. et al.,Oncology. 1991;48(4):321-6; Kobayashi, H. et al., J Clin Oncol. 1992Jan;10(1):95-101). Tn and STn formation is associated with somaticmutations in the gene Cosmc that encodes a molecular chaperon requiredfor the formation of the activate T-synthase (Ju, T. et al., Nature.2005 Oct 27;437(7063):1252; Ju, T. et al., Cancer Res. 2008 Mar15;68(6):1636-46). It can also result from increased expression of thesialyl transferase, ST6GalNAc-I (Ikehara, Y. et al., Glycobiology. 1999Nov;9(11):1213-24; Brockhausen, I. et al., Biol Chem. 2001Feb;382(2):219-32). De-novo expression of STn can modulate carcinomacells, change the malignant phenotype, and lead to more aggressive cellbehaviors (Pinho, S. et al., Cancer Lett. 2007 May 8;249(2):157-70).Although STn is highly expressed in malignant tissues, low levels arealso found on healthy human cells (Jass, J.R. et al., J Pathol. 1995Jun;176(2):143-9; Kirkeby, S. et al., Arch Oral Biol. 2010Nov;55(11):830-41). STn alone has attracted attention as a target forcancer detection and therapy (Cheever, M.A. et al., Clin Cancer Res.2009 Sep 1;15(17):5323-37). STn is also present in mucins associatedwith cancer stem cells (Engelmann et al., Cancer research, 2008, 68,2419-2426) and STn is implicated in immune supression (Carrascal, M.A.,et al., Molecular Oncology. 2014. 8(3): 753-65).

In addition to the presence of STn, other glycosylation changes havebeen described in cancer. One of them involves Neu5Gc.N-acetylneuraminic acid (Neu5Ac) and Neu5Gc are the two major sialicacids on mammalian cell surfaces. Neu5Ac and Neu5Gc differ only in thatNeu5Gc includes an additional oxygen atom associated with chemical groupattached to carbon 5. Due to the loss of a functional gene, humans canonly synthesize sialic acid in the form of Neu5Ac, but not Neu5Gc.However Neu5Gc can be metabolically incorporated into humans fromanimal-derived dietary sources such as red meats (Tangvoranuntakul, P.et al., Proc Natl Acad Sci U S A. 2003 Oct 14; 100(21):12045-50; Nguyen,D.H. et al., J Immunol. 2005 Jul 1; 175(1):228-36; US7,682,794,US8,084,219, US2012/0142903, WO2010030666 and WO2010030666). Neu5Gc issignificantly abundant among human tumors (Higashi, H. et al., CancerRes. 1985 Aug; 45(8):3796-802; Miyoshi I. et al., Mol Immunol. 1986. 23:631-638; Hirabayashi, Y. et al., Jpn J Cancer Res. 1987. 78: 614-620;Kawachi. S, et al., Int Arch Allergy Appl Immunol. 1988. 85: 381-383;Devine, P.L. et al., Cancer Res. 1991. 51: 5826-5836; Malykh, Y.N. etal, Biochimie. 2001. 83: 623-634 and Inoue, S. et al., 2010.Glycobiology. 20(6): 752-762) and remarkably low in normal humantissues, which had been overlooked for several decades (Diaz, S.L. etal., PLoS One. 2009. 4: e4241; Tangvoranuntakul, P. et al., Proc NatlAcad Sci U S A. 2003. 100: 12045-12050; Varki, A. et al., Glycoconj J.2009. 26: 231-245). The increased metabolic accumulation of diet-derivedNeu5Gc in cancer tissue compared to healthy human tissues is likelyexplained by at least three factors: rapid growth with underproductionof competing endogenous Neu5Ac, enhanced macropinocytosis induced bygrowth factors (Dharmawardhane, S. et al., Mol Biol Cell. 2000Oct;11(10):3341-52; Simonsen, A. et al., Curr Opin Cell Biol. 2001Aug;13(4):485-92; Johannes, L. et al., Traffic. 2002 Jul;3(7):443-51;Amyere, M. et al., Int J Med Microbiol. 2002 Feb;291(6-7):487-94), andthe upregulation of gene expression of the lysosomal sialic acidtransporter gene sialin by hypoxia (Yin, J. et al., Cancer Res. 2006 Mar15;66(6):2937-45). In addition, all humans tested to date include apolyclonal antibody reservoir against non-human Neu5Gc, which makes itthe first example of a xeno-autoantigen (Padler-Karavani, V. et al.,Glycobiology. 2008 Oct;18(10):818-30; Varki, N.M. et al., Annu RevPathol. 2011;6:365-93). The accumulation of dietary Neu5Gc in malignanttumors in the face of an anti-Neu5Gc response was shown to facilitatetumor progression by inducing a low-grade chronic inflammation (Hedlund,M. et al., Proc Natl Acad Sci U S A. 2008 Dec 2;105(48):18936-41). Thus,Neu5Gc containing glycan epitopes on human tumors represent a valuablepossibility for drug targeting. A recent study suggests the existence ofantibodies against Neu5Gc-containing STn (GcSTn), but notNeu5Ac-STn(AcSTn), in cancer patients and explores their potential as aspecific biomarker for cancer detection (Padler-Karavani, V. et al.,Cancer Res. 2011 May 1;71(9):3352-63).

There remains a need in the art for therapeutic antibodies capable ofbinding glycans, including glycans associated with disease and diseasedcells and tissues. Further, there remains a need for better methods todevelop such antibodies and methods of using these antibodies to targetdiseased cells and tissues. The present disclosure meets these needs byproviding related compounds and methods.

SUMMARY OF THE INVENTION

In some embodiments, the present disclosure provides an antibody havinga heavy chain variable domain (VH) with a CDR-H3 complementaritydetermining region having at least 50% amino acid sequence identity toan amino sequence selected from the group consisting of SEQ ID NOs:114-120, 140, and 141. The VH may include a CDR-H1 having at least 50%sequence identity to an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 105, 106, and 136 and a CDR-H2 having at least60% sequence identity to an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 107-113, and 137-139.

In some embodiments, antibodies include a light chain variable domain(VL) with a CDR-L3 having at least 50% amino acid sequence identity toan amino sequence selected from the group consisting of SEQ ID NOs: 89,91, 93, 95-98, 101-103, 133-135, and 148. The VL may include a CDR-L1having at least 50% sequence identity to an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 121-129, and 142-146 and aCDR-L2 having at least 50% sequence identity to an amino acid sequenceselected from the group consisting of SEQ ID NOs: 77, 79-81, 83-86, 88,130-132, and 147. The antibody may include at least one human frameworkregion having an amino acid sequence with at least 70% sequence identityto an amino acid sequence selected from the group consisting of SEQ IDNOs: 206-216.

In some embodiments, antibodies include a VH having at least 70%sequence identity to an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 220-224, 230-234, 237-241, 249-253, and256-260. Antibodies may include a VL having at least 70% sequenceidentity to an amino acid sequence selected from the group consisting ofSEQ ID NOs: 217-219, 225-229, 235, 236, 242-248, 254, and 255. The VHmay have at least 95% sequence identity to an amino acid sequenceselected from the group consisting of SEQ ID NOs: 220-224 and the VL mayhave at least 95% sequence identity to an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 217-219. The VH may have atleast 95% sequence identity to an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 237-241 and the VL may have at least 95%sequence identity to an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 235 and 236. Antibodies may be an isotypeselected from the group consisting IgG1, IgG2a, IgG2b, IgG2c, IgG3, andIgG4. Antibodies may be a human or humanized antibody. The antibodiesmay be a human IgG1 antibody.

In some embodiments, the present disclosure provides a construct thatmay encode a described antibody. Further provided are cells that mayinclude the construct. The construct may be included in a vector. Insome embodiments, antibodies are provided that are produced from cellsincluding a construct encoding an antibody of the present disclosure.

In some embodiments, antibodies are provided that bind tocell-associated STn with a half maximal effective concentration (EC50)of from about 0.01 nM to about 30 nM.

In some embodiments, antibodies may be conjugated to a therapeuticagent. The therapeutic agent may be a cytotoxic agent selected from thegroup consisting of monomethyl auristatin E (MMAE) and monomethylauristatin F (MMAF). The antibody may be capable of killing anSTn-associated cell with a half-maximal inhibitory concentration (IC50)of from about 0.1 nM to about 20 nM.

In some embodiments, methods of treating cancer are provided thatinclude administering disclosed antibodies. The cancer may include atleast one tumor. The volume of the tumor may be reduced by thetreatment. The reduction in tumor size may be at least 20%. The tumormay include at least one tumor cell, which may include at least onetumor-associated carbohydrate antigen (TACA). The TACA may includesialyl(α2,6)N-acetylgalactosamine (STn). The cancer may include one ormore of breast cancer, colon cancer, pancreatic cancer, lung cancer,cervical cancer, ovarian cancer, stomach cancer, prostate cancer, andliver cancer. The antibodies may be administered in combination with achemotherapeutic agent and/or therapeutic antibody. The chemotherapeuticagent may be selected from at least one of fluoropyrimidine,oxaliplatin, and irinotecan. The therapeutic antibody may be selectedfrom at least one of bevacizumab and anti-epidermal growth factorreceptor (EGFR) antibody. The antibodies may be administered at a doseof from about 0.1 mg/kg to about 30 mg/kg. The dose may be from about2.5 mg/kg to about 5 mg/kg. The antibodies may be detectable in at leastone subject sample obtained from about 1 day after treatment to about 1month after treatment. The antibodies may be conjugated with MMAE. Thedrug to antibody ratio (DAR) of the MMAE to the antibody in the samplemay change by less than 50% in the at least one subject sample.

In some embodiments, a method of screening a cell or sample for thepresence of at least one TACA is provided that includes contacting thecell or sample with a disclosed antibody. The at least one TACA mayinclude STn. The sample may be a biological sample. The biologicalsample may be obtained from a subject. The subject may have or besuspected of having cancer. The biological sample may include one ormore of a cell, a tissue, a tissue section, and a body fluid. Theantibody may include a detectable label. The antibody may be detectedusing a detection agent. The detection agent may be a secondaryantibody. The secondary antibody may include a detectable label. Themethod may be used for diagnosing cancer in a subject. The method may bepart of a companion diagnostic. The companion diagnostic may be used forone or more of stratifying cancer severity, stratifying cancer risk,selecting a subject for a clinical trial, developing a therapeuticregimen, modulating a therapeutic regimen, increasing treatment safety,and modulating treatment effectiveness. The method may include the useof a protein array. The protein array may include one or more antibodiesconfigured to bind one or more proteins present in the sample.

In some embodiments, the present disclosure provides a kit for carryingout described methods. The kit may include a described antibody. The kitmay include a secondary antibody. The secondary antibody may include adetectable label.

In some embodiments, the present disclosure provides a composition thatincludes one or more of the antibodies described. The composition mayinclude at least one excipient. The composition may include apharmaceutically acceptable excipient. The composition may include anantibody-coated agent. The antibody-coated agent may include one or moreof a particle, a nanoparticle, a protein, a fusion-protein, a lipid, aliposome, and a cell. The antibody may be an antibody fragment. Theantibody fragment may be selected from one or more of a Fab fragment anda single chain Fv.

In some embodiments, the present disclosure provides a modified cellhaving a synthetic construct. The synthetic construct may encode afactor that modulates cellular STn levels. The factor may include atleast one factor involved in STn synthesis The factor may be selectedfrom at least one of(Alpha-N-Acetyl-Neuraminyl-2,3-Beta-Galactosyl-1,3)-N-Acetylgalactosaminide,Alpha-2,6-Sialyltransferase I (ST6GalNAc I), T-synthase, and Core 1Beta3-Galactosyltransferase-Specific Molecular Chaperone (COSMC). Themodified cell may include elevated STn levels when compared with atleast one unmodified cell. The factor may reduce expression ofST6GalNAC. The factor may be an inhibitory ribonucleic acid (RNA)molecule. The modified cell may be a modified ovarian tumor cell. Themodified ovarian tumor cell may be selected from one or more of a SKOV3cell, an OVCAR3 cell, an OVCAR4 cell, a BRCA1 mutant tumor cell, and anon-BRCA1 mutant tumor cell.

In some embodiments, a method of characterizing antibody binding isprovided. The method may include contacting a glycan array with theantibody. The glycan array may include a plurality of glycans. Theplurality of glycans may include a panel of glycans consisting of one ormore of each of Neu5Acα6GalNAcαO(CH2)2CH2NH2;Neu5Gcα6GalNAcαO(CH2)2CH2NH2; Neu5Acα6Galβ4GlcNAcβO(CH2)2CH2NH2;Neu5Gcα6Galβ4GlcNAcβO(CH2)2CH2NH2; Neu5Acα6Galβ4GlcβO(CH2)2CH2NH2;Neu5Gcα6Galβ4GlcβO(CH2)2CH2NH2; Neu5Acα6GalβO(CH2)2CH2NH2;Neu5Gcα6GalβO(CH2)2CH2NH2; GalNAcαO(CH2)2CH2NH2;Galβ3GalNAcβO(CH2)2CH2NH2; Gal3βGalNAcαO(CH2)2CH2NH2;Neu5Acα3Galβ1-3GalNAcαO(CH2)2CH2NH2; andNeu5Gcα3Galβ1-3GalNAcαO(CH2)2CH2NH2. Each of the plurality of glycansmay be part of a neoglycolipid probe.

BRIEF DESCRIPTION OF THE FIGURES

The foregoing and other objects, features and advantages will beapparent from the following description of particular embodiments of theinvention, as illustrated in the accompanying drawings in which likereference characters refer to the same parts throughout the differentviews. The drawings are not necessarily to scale, emphasis instead beingplaced upon illustrating the principles of various embodiments of theinvention.

FIGS. 1A-1D are diagrams depicting α2,6-sialylated N-acetylgalactosamine(STn) and indicating putative epitopes involved in anti-STn antibodybinding. The largest ellipse in each diagram indicates the specificregion of STn targeted by each of 4 antibody groups. These groupsinclude Group 1 antibodies (binding to the large elliptical regionindicated in FIG. 1A), Group 2 antibodies (binding to the largeelliptical region indicated in FIG. 1B), Group 3 antibodies (binding tothe large elliptical region indicated in FIG. 1C) and Group 4 antibodies(binding to the large elliptical region indicated in FIG. 1D).

FIG. 2 is a schematic of a variable domain.

DETAILED DESCRIPTION Introduction

According to the present invention are antibodies specific for or whichinteract with epitopes that include carbohydrate groups referred toherein as glycans. Some glycan-interacting antibodies described hereinmay be used as biotherapeutics. Other embodiments provide methods forgenerating such glycan-interacting antibodies.

In nature, STns may be sialylated with N-acetylneuraminic acid (Neu5Ac)or N-glycolylneuraminic acid (Neu5Gc). Glycan-interacting antibodiesaccording to the present invention may be directed to glycans having anySTns (pan-STn antibodies), glycans having STns that include Neu5Acspecifically (AcSTn) or glycans having STns that include Neu5Gcspecifically (GcSTn). In some embodiments, glycan-interacting antibodiesof the present invention target cancer-related glycan antigens, such asα2,6-sialylated N-acetylgalactosamine (STn).

In some embodiments, the present disclosure provides methods ofproducing glycan-interacting antibodies. Such methods may include theuse of mice for generating an immune response to one or more antigens,including STn (e.g. AcSTn and/or GcSTn). As described herein, a numberof methods may be utilized in order to manipulate the resultingantibodies produced through mouse immunization. Such methods may includevarying the strain and/or gender of the mice being immunized, varyingthe antigen used, varying the type and dose of adjuvant included inantigen administration and time course of immunization before initiationof hybridoma fusion.

In some embodiments, the present disclosure provides methods foreliminating cancer stem cells using glycan-interacting antibodies. Inother embodiments, the present invention provides methods for treatingcancer in a subject by eliminating cancer stem cells usingglycan-interacting antibodies. In some aspects, glycan-interactingantibodies may be used alone. In other aspects, glycan-interactingantibodies are used in combination with chemotherapeutic agents.

Further provided are optimized, humanized and conjugated forms ofglycan-interacting antibodies disclosed herein. Additionally, kits,assays and reagents including antibodies and/or methods of the presentinvention are presented.

Definitions

Adjacent: As used herein, the term “adjacent” refers to something thatis adjoining, neighboring or next to a given entity. In someembodiments, “adjacent residues” are sugar residues within a glycanchain that are linked to one another. In some embodiments, “adjacentglycans” are glycan chains that next to each other either in directcontact or within close proximity and without another glycan in betweenthe two.

Administered in combination: As used herein, the term “administered incombination” or “combined administration” means that a subject issimultaneously exposed to two or more agents administered at the sametime or within an interval of time such that the subject is at somepoint in time simultaneously exposed to both and/or such that there maybe an overlap in the effect of each agent on the patient. In someembodiments, at least one dose of one or more agents is administeredwithin about 24 hours, 12 hours, 6 hours, 3 hours, 1 hour, 30 minutes,15 minutes, 10 minutes, 5 minutes, or 1 minute of at least one dose ofone or more other agents. In some embodiments, administration occurs inoverlapping dosage regimens. As used herein, the term “dosage regimen”refers to a plurality of doses spaced apart in time. Such doses mayoccur at regular intervals or may include one or more hiatus inadministration. In some embodiments, the administration of individualdoses of one or more glycan-interacting antibodies, as described herein,are spaced sufficiently closely together such that a combinatorial(e.g., a synergistic) effect is achieved.

Amino acid: As used herein, the terms “amino acid” and “amino acids”refer to all naturally occurring L-alpha-amino acids as well asnon-naturally occurring amino acids. Amino acids are identified byeither the one-letter or three-letter designations as follows: asparticacid (Asp:D), isoleucine (Ile:I), threonine (Thr:T), leucine (Leu:L),serine (Ser:S), tyrosine (Tyr:Y), glutamic acid (Glu:E), phenylalanine(Phe:F), proline (Pro:P), histidine (His:H), glycine (Gly:G), lysine(Lys:K), alanine (Ala:A), arginine (Arg:R), cysteine (Cys:C), tryptophan(Trp:W), valine (Val:V), glutamine (Gln:Q) methionine (Met:M),asparagine (Asn:N), where the amino acid is listed first followedparenthetically by the three and one letter codes, respectively.

Animal: As used herein, the term “animal” refers to any member of theanimal kingdom. In some embodiments, “animal” refers to humans at anystage of development. In some embodiments, “animal” refers to non-humananimals at any stage of development. In certain embodiments, thenon-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit,a monkey, a dog, a cat, a sheep, cattle, a primate, or a pig). In someembodiments, animals include, but are not limited to, mammals, birds,reptiles, amphibians, fish, and worms. In some embodiments, the animalis a transgenic animal, genetically-engineered animal, or a clone.

Antibody: As used herein, the term “antibody” is used in the broadestsense and specifically covers various embodiments including, but notlimited to monoclonal antibodies, polyclonal antibodies, multispecificantibodies (e.g. bispecific antibodies formed from at least two intactantibodies), and antibody fragments such as diabodies so long as theyexhibit a desired biological activity. Antibodies are primarilyamino-acid based molecules but may also include one or moremodifications such as with sugar moieties.

Antibody fragment: As used herein, the term “antibody fragment” refersto a portion of an intact antibody, preferably including an antigenbinding region thereof. Examples of antibody fragments include Fab,Fab′, F(ab′)₂, and Fv fragments; diabodies; linear antibodies;single-chain antibody molecules; and multispecific antibodies formedfrom antibody fragments. Papain digestion of antibodies produces twoidentical antigen-binding fragments, called “Fab” fragments, each with asingle antigen-binding site. Also produced is a residual “Fc” fragment,whose name reflects its ability to crystallize readily. Pepsin treatmentyields an F(ab′)₂ fragment that has two antigen-binding sites and isstill capable of cross-linking antigen. Glycan-interacting antibodiesmay include one or more of these fragments. For the purposes herein, anantibody may include a heavy and light variable domain as well as an Fcregion.

Antigen-binding region: As used herein, the term “antigen-bindingregion” refers to the portion of an antibody, antibody fragment, orrelated molecule that directly interacts with a target molecule orepitope. Antigen-binding regions typically include a variable domainpair, as in the Fab region of an antibody or as linked together in ascFv.

Approximately: As used herein, the term “approximately” or “about,” asapplied to one or more values of interest, refers to a value that issimilar to a stated reference value. In certain embodiments, the term“approximately” or “about” refers to a range of values that fall within25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than orless than) of the stated reference value unless otherwise stated orotherwise evident from the context (except where such number wouldexceed 100% of a possible value).

Associated with: As used herein, the terms “associated with,”“conjugated,” “linked,” “attached,” and “tethered,” when used withrespect to two or more moieties, means that the moieties are physicallyassociated or connected with one another, either directly or via one ormore additional moieties that serves as a linking agent, to form astructure that is sufficiently stable so that the moieties remainphysically associated under the conditions in which the structure isused, e.g., physiological conditions. An “association” need not bestrictly through direct covalent chemical bonding. It may also suggestionic or hydrogen bonding or a hybridization based connectivitysufficiently stable such that the “associated” entities remainphysically associated.

Bifunctional: As used herein, the term “bifunctional” refers to anysubstance, molecule or moiety which is capable of or maintains at leasttwo functions. The functions may affect the same outcome or a differentoutcome. The structure that produces the function may be the same ordifferent.

Biomolecule: As used herein, the term “biomolecule” is any naturalmolecule which is amino acid-based, nucleic acid-based,carbohydrate-based or lipid-based, and the like.

Bispecific antibody: As used herein, the term “bispecific antibody”refers to an antibody capable of binding two different antigens. Suchantibodies typically include regions from at least two differentantibodies. Bispecific antibodies may include any of those described inRiethmuller, G. 2012. Cancer Immunity. 12:12-18, Marvin, J.S. et al.,2005. Acta Pharmacologica Sinica. 26(6):649-58 and Schaefer, W. et al.,2011. PNAS. 108(27):11187-92, the contents of each of which are hereinincorporated by reference in their entirety.

Branch: As used herein, the term “branch” refers to an entity, moiety orappendage that is linked or extends out from a main entity or source. Insome embodiments, a “branch chain” or “branching chain” includes one ormore residues (including, but not limted to sugar residues) that extendfrom a parent chain. As used herein, a “parent chain” is used to referto a chain of residues (including, but not limited to sugar residues)from which a branching chain is linked. In the case of a glycan withmultiple branches, the parent chain may also refer to the source chainfrom which all such branches are directly or indirectly attached. In thecase of a polysaccharide having a chain of hexose residues, parent chainlinkages typically occur between carbons 1 and 4 of adjacent residueswhile branching chains are attached to a parent chain through a linkagebetween carbon 1 of the branching residue and carbon 3 of the parentresidue from which the branch extends. As used herein, the term“branching residue” refers to the residue attached to the parent chainin a branching chain.

Cancer stem cells: As used herein, cancer stem cells (CSCs) refer to asubset of tumor cells that have the ability to self-renew. CSCs may beable to regenerate diverse cell types. In some cases, these cells aredifficult or impossible to remove through surgical or chemical treatmentof a tumor.

Compound: As used herein, the term “compound,” refers to a distinctchemical entity. In some embodiments, a particular compound may exist inone or more isomeric or isotopic forms (including, but not limited tostereoisomers, geometric isomers and isotopes). In some embodiments, acompound is provided or utilized in only a single such form. In someembodiments, a compound is provided or utilized as a mixture of two ormore such forms (including, but not limited to a racemic mixture ofstereoisomers). Those of skill in the art appreciate that some compoundsexist in different such forms, show different properties and/oractivities (including, but not limited to biological activities). Insuch cases it is within the ordinary skill of those in the art to selector avoid particular forms of the compound for use in accordance with thepresent invention. For example, compounds that contain asymmetricallysubstituted carbon atoms can be isolated in optically active or racemicforms. Methods on how to prepare optically active forms from opticallyactive starting materials are known in the art, such as by resolution ofracemic mixtures or by stereoselective synthesis.

Cyclic or Cyclized: As used herein, the term “cyclic” refers to thepresence of a continuous loop. Cyclic molecules need not be circular,only joined to form an unbroken chain of subunits.

Cytidine monphosphate-N-acetylneuraminic acid hydroxylase: As usedherein, the term “cytidine monophosphate-N-acetylneuraminic acidhydroxylase” or “CMAH” refers to an enzyme, absent in humans, butpresent in most other mammals (including, but not limited to mice, pigsand chimpanzees) that catalyzes the formation of N-glycolylneuraminicacid from N-acetylneuraminic acid. The absence of the enzyme in humansis due to a frameshift mutation resulting in the premature terminationof the CMAH transcript and the production of a non-functional protein.

Cytotoxic: As used herein, the term “cytotoxic” is used to refer to anagent that kills or causes injurious, toxic, or deadly effects on a cell(e.g., a mammalian cell (e.g., a human cell)), bacterium, virus, fungus,protozoan, parasite, prion, or a combination thereof.

Delivery: As used herein, “delivery” refers to the act or manner oftransporting a compound, substance, entity, moiety, cargo or payload toan intended destination.

Delivery Agent: As used herein, “delivery agent” refers to any substancewhich facilitates, at least in part, the in vivo delivery of a compound,substance, entity, moiety, cargo or payload.

Detectable label: As used herein, “detectable label” refers to one ormore markers, signals, or moieties which are attached, incorporated orassociated with another entity, which markers, signals or moieties arereadily detected by methods known in the art including radiography,fluorescence, chemiluminescence, enzymatic activity, absorbance and thelike. Detectable labels include radioisotopes, fluorophores,chromophores, enzymes, dyes, metal ions, ligands such as biotin, avidin,streptavidin and haptens, quantum dots, and the like. Detectable labelsmay be located at any position in the entity with which they areattached, incorporated or associated. For example, when attached,incorporated in or associated with a peptide or protein, they may bewithin the amino acids, the peptides, or proteins, or located at the N-or C- termini.

Display library: As used herein, the term “display library” refers to atool used in scientific discovery to identify biomolecular interactions.Different variations of display libraries exist that include theutilization of bacteriophages, yeast and ribosomes. In each case,proteins within a given library (also referred to herein as “librarymembers”) are linked (physically or through association with a host) tothe nucleic acid which encodes the protein. When a target molecule isincubated with the members of a display library, any library membersthat bind to the target may be isolated and the sequences encoding thebound protein may be determined through analysis of the linked nucleicacid. In some embodiments, display libraries are “phage displaylibraries” wherein the display library is made up of bacteriophage viralparticles (also referred to herein as “phage particles”) wherein nucleicacids have been incorporated into the phage genome resulting in theproduction of viral coat proteins that are fused to proteins encoded bythe nucleic acids that have been introduced. Such fused proteins are“displayed” on the outer surface of the assembled phage particles wherethey may interact with a given target.

Distal: As used herein, the term “distal” means situated away from thecenter or away from a point or region of interest.

Engineered: As used herein, embodiments of the invention are“engineered” when they are designed to have a feature or property,whether structural or chemical, that varies from a starting point, wildtype or native molecule. Thus, engineered agents or entities are thosewhose design and/or production include an act of the hand of man.

Epitope: As used herein, an “epitope” refers to a surface or region on amolecule that is capable of interacting with components of the immunesystem, including, but not limited to antibodies. In some embodiments,an epitope may include a target site. Epitopes may include a region onan antigen or between two or more antigens that is specificallyrecognized and bound by a corresponding antibody. Some epitopes mayinclude one or more sugar residues along one or more glycan. Suchepitopes may include 1, 2, 3, 4, 5, 6, 7, 8, 9 or at least 10 sugarresidues. Epitopes may also include one or more regions of interactionbetween entities. In some embodiments, epitopes may include a junctionbetween two sugar residues, between a branching chain and a parent chainor between a glycan and a protein.

Ether bond: As used herein, an “ether bond” refers to a chemical bondthat includes an oxygen bonded between two carbon atoms. In someembodiments, ether bonds link sugar residues to other entities,including, but not limited to other sugar residues to form a glycanchain. Such bonds are also referred to as “glycosidic bonds” or“glycosidic linkages”. In the context of at least one sugar residue, theterms “link” and/or “linkage” are also used herein when referring to aglycosidic linkage. In some embodiments, linkages may link glycans toother entities, including, but not limited to proteins, lipids,phospholipids and sphingolipids. In some embodiments, sugar residues maybe linked to protein, typically forming a link between a sugar residueand an amino acid residue. Such amino acid residues include serine andthreonine. In some embodiments, ether bonds link glycans to a glycanarray through a carbohydrate linker that participates in bond formation.Glycosidic linkages may differ in their stereochemical properties. Insome embodiments, alpha oriented glycosidic linkages (also referred toherein as “alpha linkages”) result in an axial orientation between thebonded oxygen of the ether bond and the cyclohexane ring of the sugarreside. In some embodiments, beta oriented glycosidic linkages (alsoreferred to herein as “beta linkages”) result in an equatorialorientation between the bonded oxygen of the ether bond and thecyclohexane ring of the sugar residue.

Expression: As used herein, “expression” of a nucleic acid sequencerefers to one or more of the following events: (1) production of an RNAtemplate from a DNA sequence (e.g., by transcription); (2) processing ofan RNA transcript (e.g., by splicing, editing, 5′ cap formation, and/or3′ end processing); (3) translation of an RNA into a polypeptide orprotein; (4) folding of a polypeptide or protein; and (5)post-translational modification of a polypeptide or protein.

Feature: As used herein, a “feature” refers to a characteristic, aproperty, or a distinctive element.

Formulation: As used herein, a “formulation” refers to a material ormixture prepared according to a formula and which may include at leastone antibody, compound, substance, entity, moiety, cargo or payload anda delivery agent, carrier or excipient.

Functional: As used herein, a “functional” biological molecule is abiological entity with a structure and in a form in which it exhibits aproperty and/or activity by which it is characterized. As used herein, a“functional group” or “chemical group” refers to a characteristic groupof atoms or chemical bonds that are part of a larger molecule. In someembodiments, functional groups may be associated with differentmolecules, but may participate in similar chemical reactions regardlessof the molecule of which they are a part. Common functional groupsinclude, but are not limited to carboxyl groups (-COOH), acetyl groups(-COH), amino groups (—NH₂), methyl groups (—CH₃), sulfate groups(—SO₃H) and acyl groups. In some embodiments, the addition of one ormore functional group to a molecule may be conveyed using terms thatmodify the name of the functional group with the ending “-ylated”, e.g.,acetylated, methylated and sulfated.

Glycan: As used herein, the terms “glycan”, “oligosaccharide” and“polysaccharide” are used interchangeably and refer to polymers made upof sugar monomers, typically joined by glycosidic bonds also referred toherein as linkages. In some embodiments, the terms “glycan”,“oligosaccharide” and “polysaccharide” may be used to refer to thecarbohydrate portion of a glycoconjugate (e.g., glycoprotein, glycolipidor proteoglycan).

Glycan chain: As used herein, the term “glycan chain” refers to a sugarpolymer that includes two or more sugars. In some embodiments, glycanchains are covalently linked to proteins through serine or threonineresidues on the protein.

Glycan-rich composition: As used herein, the term “glycan-richcomposition” refers to a mixture that includes a large percentage ofglycans. In some embodiments, glycans within a glycan-rich compositionmay make up from about 1% to about 10%, from about 5% to about 15%, fromabout 20% to about 40%, from about 30% to about 50%, from about 60% toabout 80%, from about 70% to about 90% or at least 100% of the totalweight of the composition.

Glycosidic bond: As used herein, the term “glycosidic bond” refers to acovalent bond formed between a carbohydrate and another chemical group.In some embodiments, glycosidic bonds are formed between the reducingend of one sugar molecule and the non-reducing end of a second sugarmolecule or polysaccharide chain. Such glycosidic bonds are also knownas O-glycosidic bonds due to the oxygen (or ether bond) between thejoined sugars. In some embodiments, a glycosidic bond between two sugarsor between a sugar and a linker may also be referred to as a “linkage”.

In vitro: As used herein, the term “in vitro” refers to events thatoccur in an artificial environment, e.g., in a test tube or reactionvessel, in cell culture, in a Petri dish, etc., rather than within anorganism (e.g., animal, plant, or microbe).

In vivo: As used herein, the term “in vivo” refers to events that occurwithin an organism (e.g., animal, plant, or microbe or cell or tissuethereof).

Isolated: As used herein, the term “isolated” is synonymous with“separated”, but carries with it the inference separation was carriedout by the hand of man. In one embodiment, an isolated substance orentity is one that has been separated from at least some of thecomponents with which it was previously associated (whether in nature orin an experimental setting). Isolated substances may have varying levelsof purity in reference to the substances from which they have beenassociated. Isolated substances and/or entities may be separated from atleast about 10%, about 20%, about 30%, about 40%, about 50%, about 60%,about 70%, about 80%, about 90%, or more of the other components withwhich they were initially associated. In some embodiments, isolatedagents are more than about 80%, about 85%, about 90%, about 91%, about92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%,about 99%, or more than about 99% pure. As used herein, a substance is“pure” if it is substantially free of other components.

Kit: As used herein, the term “kit” refers to a set that includes one ormore components adapted for a cooperative purpose and instructions foruse thereof.

Knockout: As used herein, the term “knockout” refers to an organismwherein an existing gene has been inactivated through a process thattypically involves the hand of man. In a knockout organism, a gene thathas been inactivated is said to have been “knocked out”. In someembodiments, the knocked out gene may be inactivated through theinsertion of a nucleotide sequence into the gene or through replacementof the gene entirely.

Linker: As used herein, a “linker” refers to a moiety that connects twoor more domains, moieties or entities. In one embodiment, a linker mayinclude 10, 11, 12, 13, 14, 15 or more atoms. In a further embodiment, alinker may include a group of atoms, e.g., 10-1,000 atoms. Such atoms orgroups thereof may include, but are not limited to, carbon, amino,alkylamino, oxygen, sulfur, sulfoxide, sulfonyl, carbonyl, and imine. Insome embodiments, the linker may include an amino acid, peptide,polypeptide or protein. In some embodiments, a moiety bound by a linkermay include, but is not limited to an atom, a chemical group, anucleoside, a nucleotide, a nucleobase, a sugar, a nucleic acid, anamino acid, a peptide, a polypeptide, a protein, a protein complex, apayload (e.g., a therapeutic agent) or a marker (including, but notlimited to a chemical, fluorescent, radioactive or bioluminescentmarker). The linker can be used for any useful purpose, such as to formmultimers or conjugates, as well as to administer a payload, asdescribed herein. Examples of chemical groups that can be incorporatedinto the linker include, but are not limited to, alkyl, alkenyl,alkynyl, amido, amino, ether, thioether, ester, alkylene,heteroalkylene, aryl, or heterocyclyl, each of which can be optionallysubstituted, as described herein. Examples of linkers include, but arenot limited to, unsaturated alkanes, polyethylene glycols (e.g.,ethylene or propylene glycol monomeric units, e.g., diethylene glycol,dipropylene glycol, triethylene glycol, tripropylene glycol,tetraethylene glycol, or tetraethylene glycol), and dextran polymers,Other examples include, but are not limited to, cleavable moietieswithin the linker, such as, for example, a disulfide bond (—S—S—) or anazo bond (—N═N—), which can be cleaved using a reducing agent orphotolysis. Non-limiting examples of a selectively cleavable bondsinclude an amido bond which may be cleaved for example by the use oftris(2-carboxyethyl)phosphine (TCEP), or other reducing agents, and/orphotolysis, as well as an ester bond which may be cleaved for example byacidic or basic hydrolysis. In some embodiments, a linker is acarbohydrate moiety used to link glycans to a substrate, such as in aglycan array. Such carbohydrate linkers include, but are not limited to—O(CH₂) ₂CH₂HN₂ and —O(CH₂)₃NHCOCH₂ (OCH₂CH₂)₆NH₂.

mRNA: As used herein, the term “mRNA” refers to messenger RNA producedas a result of gene transcription and processing of the generatedtranscript. In some embodiments, mRNA that has left the nucleus of thecell may be extracted from a cell or set of cells and analyzed todetermine which genes have undergone transcription at a given time orunder a given set of circumstances.

Mucin: As used herein, the term “mucin” refers to a family of proteinsthat are heavily glycosylated. In some embodiments mucins are producedby the submaxillary glands and are found in saliva and mucous.

Negative selection: As used herein, the term “negative selection” refersto the selection of library members from a display library based ontheir ability to bind entities and/or components of a composition thatdo not include a target antigen. In some embodiments, negative selectionis used prior to positive selection to remove elements that might bindnon-specifically to the target.

Off-target: As used herein, “off target” refers to any unintended effecton any one or more target, gene, or cellular transcript.

Patient: As used herein, “patient” refers to a subject who may seek orbe in need of treatment, requires treatment, is receiving treatment,will receive treatment, or a subject who is under care by a trained(e.g., licensed) professional for a particular disease or condition.

Peptide: As used herein, “peptide” is a protein or polypeptide which isless than or equal to 50 amino acids long, e.g., about 5, 10, 15, 20,25, 30, 35, 40, 45, or 50 amino acids long.

Pharmaceutically acceptable: The phrase “pharmaceutically acceptable” isemployed herein to refer to those compounds, materials, compositions,and/or dosage forms which are, within the scope of sound medicaljudgment, suitable for use in contact with the tissues of human beingsand animals without excessive toxicity, irritation, allergic response,or other problem or complication, commensurate with a reasonablebenefit/risk ratio.

Pharmaceutically acceptable excipients: The phrase “pharmaceuticallyacceptable excipient,” as used herein, refers any ingredient other thanactive agents (e.g., as described herein) present in a pharmaceuticalcomposition and having the properties of being substantially nontoxicand non-inflammatory in a patient. In some embodiments, apharmaceutically acceptable excipient is a vehicle capable of suspendingor dissolving the active agent. Excipients may include, for example:antiadherents, antioxidants, binders, coatings, compression aids,disintegrants, dyes (colors), emollients, emulsifiers, fillers(diluents), film formers or coatings, flavors, fragrances, glidants(flow enhancers), lubricants, preservatives, printing inks, sorbents,suspensing or dispersing agents, sweeteners, and waters of hydration.Exemplary excipients include, but are not limited to: butylatedhydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic),calcium stearate, croscarmellose, crosslinked polyvinyl pyrrolidone,citric acid, crospovidone, cysteine, ethylcellulose, gelatin,hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose,magnesium stearate, maltitol, mannitol, methionine, methylcellulose,methyl paraben, microcrystalline cellulose, polyethylene glycol,polyvinyl pyrrolidone, povidone, pregelatinized starch, propyl paraben,retinyl palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch(corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A,vitamin E, vitamin C, and xylitol.

Pharmaceutically acceptable salts: Pharmaceutically acceptable salts ofthe compounds described herein are forms of the disclosed compoundswherein the acid or base moiety is in its salt form (e.g., as generatedby reacting a free base group with a suitable organic acid). Examples ofpharmaceutically acceptable salts include, but are not limited to,mineral or organic acid salts of basic residues such as amines; alkalior organic salts of acidic residues such as carboxylic acids; and thelike. Representative acid addition salts include acetate, adipate,alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate,borate, butyrate, camphorate, camphorsulfonate, citrate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate,hexanoate, hydrobromide, hydrochloride, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, toluenesulfonate, undecanoate, valerate salts, and thelike. Representative alkali or alkaline earth metal salts includesodium, lithium, potassium, calcium, magnesium, and the like, as well asnontoxic ammonium, quaternary ammonium, and amine cations, including,but not limited to ammonium, tetramethylammonium, tetraethylammonium,methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine,and the like. Pharmaceutically acceptable salts include the conventionalnon-toxic salts, for example, from non-toxic inorganic or organic acids.In some embodiments a pharmaceutically acceptable salt is prepared froma parent compound which contains a basic or acidic moiety byconventional chemical methods. Generally, such salts can be prepared byreacting the free acid or base forms of these compounds with astoichiometric amount of the appropriate base or acid in water or in anorganic solvent, or in a mixture of the two; generally, nonaqueous medialike ether, ethyl acetate, ethanol, isopropanol, or acetonitrile arepreferred. Lists of suitable salts are found in Remington’sPharmaceutical Sciences, 17^(th) ed., Mack Publishing Company, Easton,Pa., 1985, p. 1418, Pharmaceutical Salts: Properties, Selection, andUse, P.H. Stahl and C.G. Wermuth (eds.), Wiley-VCH, 2008, and Berge etal., Journal of Pharmaceutical Science, 66, 1-19 (1977), each of whichis incorporated herein by reference in its entirety. Pharmaceuticallyacceptable solvate: The term “pharmaceutically acceptable solvate,” asused herein, refers to a crystalline form of a compound whereinmolecules of a suitable solvent are incorporated in the crystal lattice.For example, solvates may be prepared by crystallization,recrystallization, or precipitation from a solution that includesorganic solvents, water, or a mixture thereof. Examples of suitablesolvents are ethanol, water (for example, mono-, di-, and tri-hydrates),N-methylpyrrolidinone (NMP), dimethyl sulfoxide (DMSO),N,N′-dimethylformamide (DMF), N,N′-dimethylacetamide (DMAC),1,3-dimethyl-2-imidazolidinone (DMEU),1,3-dimethyl-3,4,5,6-tetrahydro-2-(1H)-pyrimidinone (DMPU), acetonitrile(ACN), propylene glycol, ethyl acetate, benzyl alcohol, 2-pyrrolidone,benzyl benzoate, and the like. When water is the solvent, the solvate isreferred to as a “hydrate.” In some embodiments, the solventincorporated into a solvate is of a type or at a level that isphysiologically tolerable to an organism to which the solvate isadministered (e.g., in a unit dosage form of a pharmaceuticalcomposition).

Pharmacokinetic: As used herein, “pharmacokinetic” refers to any one ormore properties of a molecule or compound as it relates to thedetermination of the fate of substances administered to a livingorganism. Pharmacokinetics is divided into several areas including theextent and rate of absorption, distribution, metabolism and excretion.This is commonly referred to as ADME where: (A) Absorption is theprocess of a substance entering the blood circulation; (D) Distributionis the dispersion or dissemination of substances throughout the fluidsand tissues of the body; (M) Metabolism (or Biotransformation) is theirreversible transformation of parent compounds into daughtermetabolites; and (E) Excretion (or Elimination) refers to theelimination of the substances from the body. In rare cases, some drugsirreversibly accumulate in body tissue.

Physicochemical: As used herein, “physicochemical” means of or relatingto a physical and/or chemical property.

Positive selection: As used herein, the term “positive selection” refersto the selection of a given entity from a group of unique entities. Suchentities and groups thereof may be, for example antibodies. In somecases they may be antibody fragments or antibody fragments expressed isassociation with an agent capable of expressing such fragments (e.g.library members from a display library). Selection may be based on theability of selected entities to bind to a desired target or epitope. Insome embodiments, positive selection may be used with phage displaylibraries to identify phage particles expressing scFvs that bind to thedesired target. In other embodiments, positive selection may refer tothe selection of antibody candidates from among a pool of antibodies. Inother cases, entities may be cells, cell lines or clones as in theslection of clones during hybridoma selection. In such cases, positiveselection may refer to clonal selection based on one or more features ofantibodies (e.g. specificity for one or more desired epitopes) producedby such clones. In some cases, desired epitopes in positive selectionmethods may include STn (e.g. AcSTn and/or GcSTn).

Conversely, “negative selection,” as used herein, included the sameprinciples and examples described for positive selection, but with thedistinguishing characteristic that it is used for removal of undesiredentities from a group of unique entities.

Preventing: As used herein, the term “preventing” refers to partially orcompletely delaying onset of an infection, disease, disorder and/orcondition; partially or completely delaying onset of one or moresymptoms, features, or clinical manifestations of a particularinfection, disease, disorder, and/or condition; partially or completelydelaying onset of one or more symptoms, features, or manifestations of aparticular infection, disease, disorder, and/or condition; partially orcompletely delaying progression from an infection, a particular disease,disorder and/or condition; and/or decreasing the risk of developingpathology associated with the infection, the disease, disorder, and/orcondition.

Prodrug: The present disclosure also includes prodrugs of the compoundsdescribed herein. As used herein, “prodrugs” refer to any substance,molecule or entity which is in a form predicate for that substance,molecule or entity to act as a therapeutic upon chemical or physicalalteration. Prodrugs may by covalently bonded or sequestered in some wayand which release or are converted into the active drug moiety prior to,upon or after administered to a mammalian subject. Prodrugs can beprepared by modifying functional groups present in the compounds in sucha way that the modifications are cleaved, either in routine manipulationor in vivo, to the parent compounds. Prodrugs include compounds whereinhydroxyl, amino, sulfhydryl, or carboxyl groups are bonded to any groupthat, when administered to a mammalian subject, cleaves to form a freehydroxyl, amino, sulfhydryl, or carboxyl group respectively. Preparationand use of prodrugs is discussed in T. Higuchi and V. Stella, “Pro-drugsas Novel Delivery Systems,” Vol. 14 of the A.C.S. Symposium Series, andin Bioreversible Carriers in Drug Design, ed. Edward B. Roche, AmericanPharmaceutical Association and Pergamon Press, 1987, both of which arehereby incorporated by reference in their entirety.

Proximal: As used herein, the term “proximal” means situated nearer tothe center or to a point or region of interest.

Region of interaction: As used herein, the term “region of interaction”refers to a region along any of two or more entities where such entitiesinteract or overlap. In some embodiments, a region of interaction mayinclude one or more sugar residues along a glycan chain that contacts asecond glycan chain. In some embodiments, the glycan chains arebranching chains from the same parent chain. In some embodiments, aregion of interaction may occur between two glycan chains wherein onechain is a branching chain and the second chain is a parent chain. Inthe case of glycan chains, regions of interaction may include 1, 2, 3,4, 5, 6, 7, 8, 9 or at least 10 sugar residues. In some embodiments,regions of interaction may also occur between glycans and proteins orbetween glycans and lipids.

Residue: As used herein, the term “residue” refers to a monomerassociated with or capable of associating with a polymer. In someembodiments, residues include sugar molecules including, but not limitedto glucose, galactose, N-acetylglucosamine, N-acetylgalactosamine,sialic acids. In some embodiments, residues include amino acids.

Sample: As used herein, the term “sample” refers to an aliquot orportion taken from a source and/or provided for analysis or processing.In some embodiments, a sample is from a biological source (also referredto herein as a “biological sample”) such as a tissue, cell or componentpart (e.g. a body fluid, including but not limited to blood, plasma,serum, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid,saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid andsemen). In some embodiments, a sample may be or include a homogenate,lysate or extract prepared from a whole organism or a subset of itstissues, cells or component parts, or a fraction or portion thereof,including but not limited to, for example, plasma, serum, spinal fluid,lymph fluid, the external sections of the skin, respiratory, intestinal,and genitourinary tracts, tears, saliva, milk, blood cells, tumors,organs. In some embodiments, a sample includes a medium, such as anutrient broth or gel, which may contain cellular components, such asproteins or nucleic acid molecule. In some embodiments, a “primary”sample is an aliquot of the source. In some embodiments, a primarysample is subjected to one or more processing (e.g., separation,purification, etc.) steps to prepare a sample for analysis or other use.

Sialyl: As used herein, the prefix “sialyl” as well as the term“sialylated” describe compounds including sialic acid.

Single unit dose: As used herein, a “single unit dose” is a dose of anytherapeutic administered in one dose/at one time/single route/singlepoint of contact, i.e., single administration event. In someembodiments, a single unit dose is provided as a discrete dosage form(e.g., a tablet, capsule, patch, loaded syringe, vial, etc).

Split dose: As used herein, a “split dose” is the division of singleunit dose or total daily dose into two or more doses.

Stable: As used herein “stable” refers to a compound or entity that issufficiently robust to survive isolation to a useful degree of purityfrom a reaction mixture, and preferably capable of formulation into anefficacious therapeutic agent.

Stabilized: As used herein, the term “stabilize”, “stabilized,”“stabilized region” means to make or become stable. In some embodiments,stability is measured relative to an absolute value. In someembodiments, stability is measured relative to a reference compound orentity.

Subject: As used herein, the term “subject” or “patient” refers to anyorganism to which a composition in accordance with the invention may beadministered, e.g., for experimental, diagnostic, prophylactic, and/ortherapeutic purposes. Typical subjects include animals (e.g., mammalssuch as mice, rats, rabbits, non-human primates, and humans) and/orplants.

Submaxillary glands: As used herein, the term “submaxillary glands” or“submandibular glands” refers to mucous producing glands located beneaththe mouth floor. These glands are capable of producing mucins and insome embodiments, may be extracted from mammals as a source of mucin.

Suffering from: An individual who is “suffering from” a disease,disorder, and/or condition has been diagnosed with or displays one ormore symptoms of a disease, disorder, and/or condition.

Susceptible to: An individual who is “susceptible to” a disease,disorder, and/or condition has not been diagnosed with and/or may notexhibit symptoms of the disease, disorder, and/or condition but harborsa propensity to develop a disease or its symptoms. In some embodiments,an individual who is susceptible to a disease, disorder, and/orcondition (for example, cancer) may be characterized by one or more ofthe following: (1) a genetic mutation associated with development of thedisease, disorder, and/or condition; (2) a genetic polymorphismassociated with development of the disease, disorder, and/or condition;(3) increased and/or decreased expression and/or activity of a proteinand/or nucleic acid associated with the disease, disorder, and/orcondition; (4) habits and/or lifestyles associated with development ofthe disease, disorder, and/or condition; (5) a family history of thedisease, disorder, and/or condition; and (6) exposure to and/orinfection with a microbe associated with development of the disease,disorder, and/or condition. In some embodiments, an individual who issusceptible to a disease, disorder, and/or condition will develop thedisease, disorder, and/or condition. In some embodiments, an individualwho is susceptible to a disease, disorder, and/or condition will notdevelop the disease, disorder, and/or condition.

Synthetic: The term “synthetic” means produced, prepared, and/ormanufactured by the hand of man. Synthesis of polynucleotides orpolypeptides or other molecules of the present invention may be chemicalor enzymatic.

Target: As used herein, the term “target” refers to an object or entityto be affected by an action. In some embodiments, targets refer toantigens to be used for the development of antibodies that specificallybind the antigens.

Target screening: As used herein, the term “target screening” refers tothe use of a target substance to identify binding partners for thatsubstance.

Target site: As used herein, the term “target site” refers to a regionon or within one or more glycans, glycoproteins, biomolecules and/orbiostructures on or within a cell, the extracellular space, a tissue, anorgan and/or an organism that is recognized by a binding agent oreffector molecule (e.g., an antibody). In some embodiments, glycantarget sites may reside exclusively on one sugar residue, may be formedby two or more residues, or may include both glycan and non-glycancomponents. In some embodiments, target sites are formed between two ormore glycans or glycoproteins. In some embodiments, target sites areformed between branching chains of the same glycan or between one ormore branching chains and a parent chain.

Targeted Cells: As used herein, “targeted cells” refers to any one ormore cells of interest. The cells may be found in vitro, in vivo, insitu or in the tissue or organ of an organism. The organism may be ananimal, preferably a mammal, more preferably a human and most preferablya patient.

Terminal residue: As used herein, the term “terminal residue” refers tothe last residue in a polymeric chain. In some embodiments, terminalresidues are sugar residues located at the non-reducing end of apolysaccharide chain.

Therapeutic agent: The term “therapeutic agent” refers to any agentthat, when administered to a subject, has a therapeutic, diagnostic,and/or prophylactic effect and/or elicits a desired biological and/orpharmacological effect.

Therapeutically effective amount: As used herein, the term“therapeutically effective amount” means an amount of an agent to bedelivered (e.g., nucleic acid, drug, therapeutic agent, diagnosticagent, prophylactic agent, etc.) that is sufficient, when administeredto a subject suffering from or susceptible to an infection, disease,disorder, and/or condition, to treat, improve symptoms of, diagnose,prevent, and/or delay the onset of the infection, disease, disorder,and/or condition. In some embodiments, a therapeutically effectiveamount is provided in a single dose. In some embodiments, atherapeutically effective amount is administered in a dosage regimenthat includes a plurality of doses. Those skilled in the art willappreciate that in some embodiments, a unit dosage form may beconsidered to include a therapeutically effective amount of a particularagent or entity if it includes an amount that is effective whenadministered as part of such a dosage regimen.

Therapeutically effective outcome: As used herein, the term“therapeutically effective outcome” means an outcome that is sufficientin a subject suffering from or susceptible to an infection, disease,disorder, and/or condition, to treat, improve symptoms of, diagnose,prevent, and/or delay the onset of the infection, disease, disorder,and/or condition.

Total daily dose: As used herein, a “total daily dose” is an amountgiven or prescribed in 24 hr period. It may be administered as a singleunit dose.

Transgenic: As used herein, the term “transgenic” refers to an organismthat includes one or more genes incorporated within the organisms genomethat are not naturally found in that organism.

Treating: As used herein, the term “treating” refers to partially orcompletely alleviating, ameliorating, improving, relieving, delayingonset of, inhibiting progression of, reducing severity of, and/orreducing incidence of one or more symptoms or features of a particularinfection, disease, disorder, and/or condition. For example, “treating”cancer may refer to inhibiting survival, growth, and/or spread of atumor. Treatment may be administered to a subject who does not exhibitsigns of a disease, disorder, and/or condition and/or to a subject whoexhibits only early signs of a disease, disorder, and/or condition forthe purpose of decreasing the risk of developing pathology associatedwith the disease, disorder, and/or condition.

Variable region: As used herein, the term “variable region” or “variabledomain” refers to specific antibody domains that differ extensively insequence among antibodies and are used in the binding and specificity ofeach particular antibody for its particular antigen.

Whole IgG: As used herein, the term “whole IgG” refers to a complete IgGmolecule. In some embodiments, whole IgG molecules include regions foundnaturally in two or more other organisms.

Wild type: As used herein, the term “wild type” refers to an organismthat includes a natural genome (free from genes derived from otherorganisms).

I. Compositions of the Invention

In some embodiments, the present invention provides compounds as well ascompositions that include at least one glycan-interacting antibody.Within a glycan, monosaccharide monomers may all be the same or they maydiffer. Common monomers include, but are not limited to trioses,tetroses, pentoses, glucose, fructose, galactose, xylose, arabinose,lyxose, allose, altrose, mannose, gulose, iodose, ribose,mannoheptulose, sedoheptulose and talose. Amino sugars may also bemonomers within a glycan. Glycans including such sugars are hereinreferred to as aminoglycans. Amino sugars, as used herein, are sugarmolecules that include an amine group in place of a hydroxyl group, orin some embodiments, a sugar derived from such a sugar. Examples ofamino sugars include, but are not limited to glucosamine, galactosamine,N-acetylglucosamine, N-acetylgalactosamine, sialic acids (including, butnot limited to, N-acetylneuraminic acid and N-glycolylneuraminic acid)and L-daunosamine.

As used herein the term “glycan-interacting antibody” refers to anantibody that can interact with a glycan moiety. Such antibodies maybind to a glycan moiety alone, to multiple glycan moieties, or toepitopes that include both glycan and non-glycan components. Non-glycancomponents may include, but are not limited to proteins,protein-associated moieties (such post-translational modifications),cells, and cell-associated molecules/structures. Glycan-interactingantibodies may function to bind to, alter, activate, inhibit, stabilize,degrade and/or modulate a glycan or a glycan-associated molecule orentity. In so doing, glycan-interacting antibodies may function as atherapeutic, whether palliative, prophylactic or as an ongoing treatmentcomposition. In some embodiments, glycan-interacting antibodies mayinclude conjugates or combinations with other molecules. In someembodiments, glycan-interacting antibodies are directed toward glycanshaving one or more amino sugar. In a further embodiment, one or moreamino sugars is a sialic acid. In a further embodiment, one or moresialic acids is N-acetylneuraminic acid and/or N-glycolylneuraminicacid.

Antibodies

Glycan-interacting antibodies may include entire antibodies or fragmentsthereof. As used herein, the term “antibody” is used in the broadestsense and embraces various formats including, but not limited tomonoclonal antibodies, polyclonal antibodies, multispecific antibodies(e.g., bispecific antibodies formed from at least two intactantibodies), antibody conjugates (including, but not limited toantibody-drug conjugates), antibody variants [including, but not limitedto antibody mimetics, chimeric antibodies (e.g. antibodies with aminoacid sequences derived from more than one species), and syntheticvariants], and antibody fragments, so long as they exhibit a desiredbiological activity (e.g., binding, activating, inhibiting, stabilizing,degrading, and/or modulating one or more targets). Antibodies areprimarily amino-acid based molecules but may include one or morepost-translational or synthetic modifications. Post-translationalmodifications may include glycosylation.

As used herein, the term “antibody fragment” refers to a portion of anintact antibody or fusion-protein thereof, in some cases including atleast one antigen binding region. Examples of antibody fragments includeFab, Fab′, F(ab′)₂, Fv fragments, single-chain variable fragments(scFvs); diabodies; tri(a)bodies; linear antibodies; single-chainantibody molecules; and multispecific antibodies formed from antibodyfragments. Papain digestion of antibodies produces two identicalantigen-binding fragments, called “Fab” fragments, each with a singleantigen-binding site. Also produced is a residual “Fc” fragment, whosename reflects its ability to crystallize readily. Pepsin treatmentyields an F(ab′)₂ fragment that has two antigen-binding sites and isstill capable of cross-linking antigen. Glycan-interacting antibodiesmay include one or more of these fragments and may, for example, begenerated through enzymatic digestion of whole antibodies or throughrecombinant expression.

“Native antibodies” are usually heterotetrameric glycoproteins of about150,000 daltons, composed of two identical light (L) chains and twoidentical heavy (H) chains. Genes encoding antibody heavy and lightchains are known and segments making up each have been wellcharacterized and described (Matsuda, F. et al., 1998. The Journal ofExperimental Medicine. 188(11); 2151-62 and Li, A. et al., 2004. Blood.103(12: 4602-9, the content of each of which are herein incorporated byreference in their entirety). Each light chain is linked to a heavychain by one covalent disulfide bond, while the number of disulfidelinkages varies among the heavy chains of different immunoglobulinisotypes. Each heavy and light chain also has regularly spacedintrachain disulfide bridges. Each heavy chain has at one end a variabledomain (V_(H)) followed by a number of constant domains. Each lightchain has a variable domain at one end (V_(L)) and a constant domain atits other end; the constant domain of the light chain is aligned withthe first constant domain of the heavy chain, and the light chainvariable domain is aligned with the variable domain of the heavy chain.

As used herein, the term “variable domain” refers to specific antibodydomains found on both the antibody heavy and light chains that differextensively in sequence among antibodies and are used in the binding andspecificity of each particular antibody for its particular antigen.Variable domains include hypervariable regions. As used herein, the term“hypervariable region” refers to a region within a variable domain thatincludes amino acid residues responsible for antigen binding. The aminoacids present within the hypervariable regions determine the structureof the complementarity determining regions (CDRs) that become part ofthe antigen-binding site of the antibody. As used herein, the term “CDR”refers to a region of an antibody that includes a structure that iscomplimentary to its target antigen or epitope. Other portions of thevariable domain, not interacting with the antigen, are referred to asframework (FW) regions. The antigen-binding site (also known as theantigen combining site or paratope) includes the amino acid residuesnecessary to interact with a particular antigen. The exact residuesmaking up the antigen-binding site are typically elucidated byco-crystallography with bound antigen, however computational assessmentscan also be used based on comparisons with other antibodies (Strohl,W.R. Therapeutic Antibody Engineering. Woodhead Publishing, PhiladelphiaPA. 2012. Ch. 3, p47-54, the contents of which are herein incorporatedby reference in their entirety). Determining residues making up CDRs mayinclude the use of numbering schemes including, but not limited to,those taught by Kabat [Wu, T.T. et al., 1970, JEM, 132(2):211-50 andJohnson, G. et al., 2000, Nucleic Acids Res. 28(1): 214-8, the contentsof each of which are herein incorporated by reference in theirentirety], Chothia [Chothia and Lesk, J. Mol. Biol. 196, 901 (1987),Chothia et al., Nature 342, 877 (1989) and Al-Lazikani, B. et al., 1997,J. Mol. Biol. 273(4):927-48, the contents of each of which are hereinincorporated by reference in their entirety], Lefranc (Lefranc, M.P. etal., 2005, Immunome Res. 1:3) and Honegger (Honegger, A. and Pluckthun,A. 2001. J. Mol. Biol. 309(3):657-70, the contents of which are hereinincorporated by reference in their entirety).

VH and VL domains have three CDRs each. VL CDRs are referred to hereinas CDR-L1, CDR-L2 and CDR-L3, in order of occurance when moving from N—to C— terminus along the variable domain polypeptide. VH CDRs arereferred to herein as CDR-H1, CDR-H2 and CDR-H3, in order of occurancewhen moving from N— to C— terminus along the variable domainpolypeptide. Each of CDRs have favored canonical structures with theexception of the CDR-H3, which includes amino acid sequences that may behighly variable in sequence and length between antibodies resulting in avariety of three-dimensional structures in antigen-binding domains(Nikoloudis, D. et al., 2014. PeerJ. 2:e456). In some cases, CDR-H3s maybe analyzed among a panel of related antibodies to assess antibodydiversity. Various methods of determining CDR sequences are known in theart and may be applied to known antibody sequences (Strohl, W.R.Therapeutic Antibody Engineering. Woodhead Publishing, Philadelphia PA.2012. Ch. 3, p47-54, the contents of which are herein incorporated byreference in their entirety).

As used herein, the term “Fv” refers to an antibody fragment thatincludes the minimum fragment on an antibody needed to form a completeantigen-binding site. These regions consist of a dimer of one heavychain and one light chain variable domain in tight, non-covalentassociation. Fv fragments can be generated by proteolytic cleavage, butare largely unstable. Recombinant methods are known in the art forgenerating stable Fv fragments, typically through insertion of aflexible linker between the light chain variable domain and the heavychain variable domain [to form a single chain Fv (scFv)] or through theintroduction of a disulfide bridge between heavy and light chainvariable domains (Strohl, W.R. Therapeutic Antibody Engineering.Woodhead Publishing, Philadelphia PA. 2012. Ch. 3, p46-47, the contentsof which are herein incorporated by reference in their entirety).

Antibody “light chains” from any vertebrate species can be assigned toone of two clearly distinct types, called kappa and lambda based onamino acid sequences of their constant domains. Depending on the aminoacid sequence of the constant domain of their heavy chains, antibodiescan be assigned to different classes. There are five major classes ofintact antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these maybe further divided into subclasses (isotypes), e.g., IgG1, IgG2a, IgG2b,IgG2c, IgG3, IgG4, IgA, and IgA2.

As used herein, the term “single chain Fv” or “scFv” refers to a fusionprotein of VH and VL antibody domains, wherein these domains are linkedtogether into a single polypeptide chain by a flexible peptide linker.In some embodiments, the Fv polypeptide linker enables the scFv to formthe desired structure for antigen binding. In some embodiments, scFvsare utilized in conjunction with phage display, yeast display or otherdisplay methods where they may be expressed in association with asurface member (e.g. phage coat protein) and used in the identificationof high affinity peptides for a given antigen.

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments include a heavy chain variabledomain V_(H) connected to a light chain variable domain V_(L) in thesame polypeptide chain. By using a linker that is too short to allowpairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies are described more fully in,for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl.Acad. Sci. USA, 90:6444-6448 (1993), the contents of each of which areincorporated herein by reference in their entirety.

The term “intrabody” referes to a form of antibody that is not secretedfrom a cell in which it is produced, but instead target one or moreintracellular protein. Intrabodies may be used to affect a multitude ofcellular processes including, but not limited to intracellulartrafficking, transcription, translation, metabolic processes,proliferative signaling and cell division. In some embodiments, methodsof the present invention may include intrabody-based therapies. In somesuch embodiments, variable domain sequences and/or CDR sequencesdisclosed herein may be incorporated into one or more construct forintrabody-based therapy. In some cases, intrabodies of the invention maytarget one or more glycated intracellular protein or may modulate theinteraction between one or more glycated intracellular protein and analternative protein.

The term “chimeric antigen receptor” or “CAR” as used herein, refers toartificial receptors that are engineered to be expressed on the surfaceof immune effector cells resulting in specific targeting of such immuneeffector cells to cells expressing entities that bind with high affinityto the artificial receptors. CARs may be designed to include one or moresegments of an antibody, antibody variable domain and/or antibody CDR,such that when such CARs are expressed on immune effector cells, theimmune effector cells bind and clear any cells that are recognized bythe antibody portions of the CARs. In some cases, CARs are designed tospecifically bind cancer cells, leading to immune-regulated clearance ofthe cancer cells.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous cells (orclones), i.e., the individual antibodies making up the population areidentical and/or bind the same epitope, except for possible variantsthat may arise during production of the monoclonal antibody, suchvariants generally being present in minor amounts. In contrast topolyclonal antibody preparations that typically include differentantibodies directed against different determinants (epitopes), eachmonoclonal antibody is directed against a single determinant on theantigen

The modifier “monoclonal” indicates the character of the antibody asbeing obtained from a substantially homogeneous population ofantibodies, and is not to be construed as requiring production of theantibody by any particular method. The monoclonal antibodies hereininclude “chimeric” antibodies (immunoglobulins) in which a portion ofthe heavy and/or light chain is identical with or homologous tocorresponding sequences in antibodies derived from a particular speciesor belonging to a particular antibody class or subclass, while theremainder of the chain(s) is identical with or homologous tocorresponding sequences in antibodies derived from another species orbelonging to another antibody class or subclass, as well as fragments ofsuch antibodies.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequences derived from non-humanimmunoglobulins. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from thehypervariable region from an antibody of the recipient are replaced byresidues from the hypervariable region from an antibody of a non-humanspecies (donor antibody) such as mouse, rat, rabbit or nonhuman primatehaving the desired specificity, affinity, and capacity. Humanizedantibodies may include one or more back-mutation that include thereversion of one or more amino acids back to amino acids found in adonor antibody. Conversely, residues from donor antibodies included inhumanized antibodies may be mutated to match residues present in humanrecipient antibodies.

In some embodiments, glycan-interacting antibodies of the presentinvention may be antibody mimetics. The term “antibody mimetic” refersto any molecule which mimics the function or effect of an antibody andwhich binds specifically and with high affinity to their moleculartargets. In some embodiments, antibody mimetics may be monobodies,designed to incorporate the fibronectin type III domain (Fn3) as aprotein scaffold (US 6,673,901; US 6,348,584). In some embodiments,antibody mimetics may be those known in the art including, but are notlimited to affibody molecules, affilins, affitins, anticalins, avimers,DARPins, Fynomers and Kunitz and domain peptides. In other embodiments,antibody mimetics may include one or more non-peptide region.

As used herein, the term “antibody variant” refers to a biomoleculeresembling an antibody in structure, sequence and/or function, butincluding some differences in their amino acid sequence, composition orstructure as compared to another antibody or a native antibody.

Antibody Development

Glycan-interacting antibodies of the present invention are developed tobind antigens such as those described herein. As used herein, an“antigen” is an entity which induces or evokes an immune response in anorganism. An immune response is characterized by the reaction of thecells, tissues and/or organs of an organism to the presence of a foreignentity. Such an immune response typically leads to the production by theorganism of one or more antibodies against the foreign entity, e.g.,antigen or a portion of the antigen. In some cases, methods ofimmunization may be altered based on one or more desired immunizationoutcomes. As used here, the term “immunization outcome” refers to one ormore desired effects of immunization. Examples include high antibodytiters and/or increased antibody specificity for a target of interest.

Antigens of the invention may include glycans, glycoconjugates(including, but not limited to glycoproteins and glycolipids), peptides,polypeptides, fusion proteins, or any of the foregoing and may beconjugated or complexed to one or more separate adjuvants orheterologous proteins. In some embodiments, antigens used according tomethods of the present invention may include sialylated glycans, such asSTn. Antigens having STn may include mucins. Mucins are a family ofproteins that are heavily glycosylated. They are a component of manytumors originating from epithelial cells (Ishida, A. et al., 2008.Proteomics. 8: 3342-9, the contents of which are herein incorporated byreference in their entirety). They are highly expressed by submaxillaryglands and can be found at high levels in saliva and mucous.Animal-derived submaxillary mucins may be used as antigens to generateanti-STn antibodies in immunogenic hosts. Submaxillary mucin fromdifferent species differ in their STn content with regard to AcSTnversus GcSTn forms. Porcine submaxillary mucin (PSM) is particularlyrich in GcSTn, which makes up about 90% of total STn. STn from bovinesubmaxillary mucin (BSM) includes roughly equal percentages of GcSTn andAcSTn. Ovine submaxillary mucin (OSM) is particularly rich in AcSTn,which makes up about 90% of total STn. In some cases, solutions preparedfor immunization may be modified to include one or more of PSM, BSM andOSM depending on the desired target of antibodies resulting from suchimmunization. PSM may be used in immunizations to generate antibodies inimmunogenic hosts that are more likely to be specific for GcSTn. PSM isrich in Neu5Gc-containing mucin-type, glycoproteins that are decoratedwith GcSTn. Among the currently known sources of high Neu5Gc content isred meat; especially submaxillary glands were previously described as arich source of Neu5Gc due to the high expression of the CMAH enzyme,which catalyzes the reaction to produce the Neu5Gc precursor,CMP-Neu5Ac. In some cases, PSM may be used to prevent a pan-anti-Neu5Gcresponse and induce a more specific immune response against GcSTn. OSMmay be used in immunizations to generate antibodies in immunogenic hoststhat are more likely to be specific for AcSTn.

In one embodiment, the present invention provides a glycan-interactingantibody that is GcSTn-specific. The antibody has littlecross-reactivity to Neu5Ac-STn or Tn. The antibody can bind GcSTn buthas reduced affinity for AcSTn.

In some embodiments, antigens may be subjected to enzymatic digestionprior to immunization to modulate the resulting immune response inimmunogenic hosts. In one example, submaxillary mucins may be treatedwith trypsin or proteinase K enzymes prior to immunization. The activityof such enzymes may help to cleave off and thereby reduce the percentageand variability of non-STn epitopes. Glycan moieties may shield regionsof the peptide where they are attached from enzymatic proteolysis andthereby remain intact. Antibody titers resulting from immunizations mayhave different antibody levels depending on the type and amount ofantigen used in such immunizations. In some cases, certain antigens maybe selected for use in immunizations based on the expected titer.

As used herein, an “adjuvant” is a pharmacological or immunologicalagent that modifies the effect of other agents. Adjuvants according tothe present invention include, but are not limited chemicalcompositions, biomolecules, therapeutics, and/or therapeutic regimens.Adjuvants may include Freund’s adjuvant (complete and/or incomplete),immunostimulatory oligonucleotides [e.g. CpG oligodeoxynucleotides(ODNs)], mineral-containing compositions, bacterial ADP-ribosylatingtoxins, bioadhesives, mucoadhesives, microparticles, lipids, liposomes,muramyl peptides, N-oxidized polyethylene-piperazine derivatives,saponins and/or immune stimulating complexes (ISCOs). In someembodiments, adjuvants may include oil-in-water emulsions (e.g.sub-micron oil-in-water emulsions). Adjuvants according to the presentinvention may also include any of those disclosed in U.S. Pat.Publication No. US20120027813 and/or U.S. Pat. No. US8506966, thecontents of each of which are herein incorporated by reference in theirentirety.

Antibodies of the present invention may be polyclonal or monoclonal orrecombinant, produced by methods known in the art or as described inthis application. In some embodiments, the antibodies of the presentinvention may be labeled for purposes of detection with a detectablelabel known by one of skill in the art. The label can be a radioisotope,fluorescent compound, chemiluminescent compound, enzyme, or enzymecofactor, or any other labels known in the art. In some aspects, theantibody that binds to a desired antigen is not labeled, but may bedetected by binding of a labeled secondary antibody that specificallybinds to the primary antibody.

Antibodies of the present invention (e.g., glycan-interactingantibodies) include, but are not limited to, polyclonal, monoclonal,multispecific, human, humanized or chimeric antibodies, single chainantibodies, Fab fragments, F(ab′) fragments, fragments produced by a Fabexpression library, anti-idiotypic (anti-Id) antibodies (including,e.g., anti-Id antibodies to antibodies of the invention),intracellularly made antibodies (i.e., intrabodies), and epitope-bindingfragments of any of the above. Antibodies of the present invention(e.g., glycan-interacting antibodies) can be from any animal originincluding birds and mammals. Preferably, such antibodies are of human,murine (e.g., mouse and rat), donkey, sheep, rabbit, goat, guinea pig,camel, horse, or chicken origin. The antibodies of the present inventioncan be monospecific or multispecific (e.g., bispecific, trispecific, orof greater multispecificity). Multispecific antibodies can be specificfor different epitopes of a target antigen of the present invention, orcan be specific for both a target antigen of the present invention, anda heterologous epitope, such as a heterologous glycan, peptide or solidsupport material. (See, e.g., WO93/17715; WO92/08802; WO91/00360;WO92/05793; Tutt, A. et al., Trispecific F(ab′)3 derivatives that usecooperative signaling via the TCR/CD3 complex and CD2 to activate andredirect resting cytotoxic T cells. J Immunol. 1991 Jul 1;147(1):60-9;U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819;and Kostelny, S.A. et al., Formation of a bispecific antibody by the useof leucine zippers. J Immunol. 1992 Mar 1;148(5):1547-53).

Glycan-interacting antibodies of the present disclosure may be preparedusing well-established methods known in the art for developingmonoclonal antibodies. In one embodiment, the monoclonal antibodies areprepared using hybridoma technology (Kohler, G. et al., Continuouscultures of fused cells secreting antibody of predefined specificity.Nature. 1975 Aug 7;256(5517):495-7). For hybridoma formations, first, amouse, hamster, or other appropriate host animal, is typically immunizedwith an immunizing agent (e.g., a target antigen of the invention) toelicit lymphocytes that produce or are capable of producing antibodiesthat will specifically bind to the immunizing agent. Alternatively, thelymphocytes may be immunized in vitro. The lymphocytes are then fusedwith an immortalized cell line using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, J.W., MonoclonalAntibodies: Principles and Practice. Academic Press. 1986; 59-1031).Immortalized cell lines are usually transformed mammalian cells,particularly myeloma cells of rodent, rabbit, bovine and human origin.Usually, rat or mouse myeloma cell lines are employed. The hybridomacells may be cultured in a suitable culture medium that preferablycontains one or more substances that inhibit the growth or survival ofthe unfused, immortalized cells. For example, if the parental cells lackthe enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT orHPRT), the culture medium for the hybridomas typically will includehypoxanthine, aminopterin, and thymidine (“HAT medium”), whichsubstances prevent the growth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More preferred immortalized cell lines are murine myeloma lines,which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Manassas, Va. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of humanmonoclonal antibodies (Kozbor, D. et al., A human hybrid myeloma forproduction of human monoclonal antibodies. J Immunol. 1984Dec;133(6):3001-5; Brodeur, B. et al., Monoclonal Antibody ProductionTechniques and Applications. Marcel Dekker, Inc., New York. 1987;33:51-63).

In some embodiments, myeloma cells may be subjected to geneticmanipulation. Such manipulation may be carried out using zinc-fingernuclease (ZFN) mutagenesis as described herein. Alternatively,transfection methods known in the art may be used. NS0 myeloma cells orother mouse myeloma cell lines may be used. For example, Sp2/0-Ag14 canbe an alternative cell line for hybridoma development.

Transcription Activator-Like Effector Nucleases (TALENs)-induced geneediting provides an alternative gene knock out method. TALENs areartificial restriction enzymes generated by fusing the TAL effector DNAbinding domain to a DNA cleavage domain. Similar to ZFNs, TALENs inducedouble-strand breaks at desired loci that can be repaired by error-proneNHEJ to yield insertions/deletions at the break sites (Wood, A.J. etal., Targeted genome editing across species using ZFNs and TALENs.Science. 2011 Jul 15;333(6040):307). Cellectis Bioresearch (Cambridge,MA) provides the service of TALEN design and plasmid construction. Theculture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies. Preferably, thebinding specificity (i.e., specific immunoreactivity) of monoclonalantibodies produced by the hybridoma cells is determined byimmunoprecipitation or by an in vitro binding assay, such asradioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).Such techniques and assays are known by those skilled in the art. Thebinding specificity of the monoclonal antibody can, for example, bedetermined by Scatchard analysis (Munson, P.J. et al., Ligand: aversatile computerized approach for characterization of ligand-bindingsystems. Anal Biochem. 1980 Sep 1;107(1):220-39). In some cases,antibody specificity for regions of a given antigen may be characterizedby chemically modifying the antigens prior to assaying for antibodybinding. In one example, periodate treatment may be used to to destroythe C6 side chain of sialic acids. Assays may be conducted with andwithout periodate treatment to reveal whether or not binding inuntreated samples is sialic acid-specific. In some cases, antigenshaving 9-O-acetylated sialic acid may be subjected to mild basetreatment (e.g. with 0.1 M NaOH) to destroy 9-O-acetyl groups. Assaysmay be conducted with and without mild base treatment to reveal whetheror not binding in untreated samples depends on 9-O-acetylation of sialicacid.

After the desired hybridoma cells are identified, the clones may besubcloned by limiting dilution procedures and grown by standard methods.Suitable culture media for this purpose include, for example, Dulbecco’sModified Eagle’s Medium or RPMI-1640 medium. Alternatively, thehybridoma cells may be grown in vivo as ascites in a mammal.

Alternative methods to clone hybridomas may include those provided bykits from STEMCELL Technologies (Vancouver, BC, Canada), e.g.ClonaCell™-HY kit, containing methylcellulose-based semi-solid mediumand other media and reagents, to support the selection and growth ofhybridoma clones. However, the media in this kit contain FCS, whichprovides an exogenous source for Neu5Gc incorporation. Though themachinery for endogenous Neu5Gc synthesis is destroyed in Cmah^(-/-)hybridoma, Neu5Gc incorporated from the culture media may also pose aproblem in some cases (Bardor, M. et al., Mechanism of uptake andincorporation of the non-human sialic acid N-glycolylneuraminic acidinto human cells. J Biol Chem. 2005. 280: 4228-4237). In such instances,The culture media may be supplemented with Neu5Ac to eliminate Neu5Gcincorporation by metabolic competition (Ghaderi, D. et al., Implicationsof the presence of N-glycolylneuraminic acid in recombinant therapeuticglycoproteins. Nat Biotechnol. 2010. 28: 863-867).

The monoclonal antibodies secreted by the subclones may be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

In another embodiment, the monoclonal antibodies of the presentinvention can also be made by recombinant DNA methods, such as thosedescribed in U.S. Pat. No. 4,816,567, which is hereby incorporated byreference in its entirety. DNA encoding the monoclonal antibodies of theinvention can be readily isolated and sequenced using conventionalprocedures (e.g., by using oligonucleotide probes that are capable ofbinding specifically to genes encoding the heavy and light chains ofmurine antibodies). The hybridoma cells of the invention serve as apreferred source of DNA. Once isolated, the DNA can be placed intoexpression vectors, which are then transfected into host cells. Hostcells may include, but are not limited to HEK293 cells, HEK293T cells,simian COS cells, Chinese hamster ovary (CHO) cells, and myeloma cellsthat do not otherwise produce immunoglobulin protein, to obtain thesynthesis of monoclonal antibodies in the recombinant host cells. TheDNA also can be modified, for example, by substituting the codingsequence for human heavy and light chain constant domains in place ofthe homologous murine sequences (U.S. Pat. No. 4,816,567) or bycovalently joining to the immunoglobulin coding sequence all or part ofthe coding sequence for a non-immunoglobulin polypeptide. Such anon-immunoglobulin polypeptide can be substituted for the constantdomains of an antibody of the invention, or can be substituted for thevariable domains of one antigen-combining site of an antibody of theinvention to create a chimeric bivalent antibody.

In some embodiments, antibodies of the present invention (e.g.,glycan-interacting antibodies) may be produced by various proceduresknown by those skilled in the art. For the production of polyclonalantibodies in vivo, host animals, such as rabbits, rats, mice, cows,horses, donkeys, chickens, monkeys, sheep or goats, are immunized witheither free or carrier-coupled antigens, for example, by intraperitonealand/or intradermal injection. In some embodiments, injection materialmay be an emulsion containing about 100 µg of antigen or carrierprotein. In some embodiments, injection materials may include aglycan-rich composition such as non-human mammalian submaxillary mucinin solution. Various adjuvants can also be used to increase theimmunological response, depending on the host species. Adjuvantsinclude, but are not limited to, Freund’s (complete and incomplete),mineral gels such as aluminum hydroxide, surface active substances suchas lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,TITERMAX® (CytRx Corp, Los Angeles, CA), keyhole limpet hemocyanins,dinitrophenol, and potentially useful human adjuvants such as BCG(bacille Calmette-Guerin) and corynebacterium parvum. Such adjuvants arealso well known in the art. Several booster injections may be needed,for instance, at intervals of about two weeks, to provide a useful titerof antibody which can be detected, for example, by ELISA assay usingglycans and/or free peptide adsorbed to a solid surface. The titer ofantibodies in serum from an immunized animal can be increased byselection of antibodies, e.g., by adsorption of antigens onto a solidsupport and elution of the selected antibodies according to methods wellknown in the art.

Glycan-interacting antibodies, variants and fragments thereof may beselected and produced using high throughput methods of discovery. In oneembodiment, glycan-interacting antibodies that include syntheticantibodies, variants and fragments thereof are produced through the useof display libraries. The term “display” as used herein, refers to theexpression or “display” of proteins or peptides on the surface of agiven host. The term “library” as used herein, refers to a collection ofunique cDNA sequences and/or the proteins that are encoded by them. Alibrary may contain from as little as two unique cDNAs to hundreds ofbillions of unique cDNAs. In some embodiments, glycan-interactingantibodies that are synthetic antibodies are produced using antibodydisplay libraries or antibody fragment display libraries. The term“antibody fragment display library” as used herein, refers to a displaylibrary wherein each member encodes an antibody fragment containing atleast one variable region of an antibody. Such antibody fragments arepreferably Fab fragments, but other antibody fragments such assingle-chain variable fragments (scFvs) are contemplated as well. In anFab antibody fragment library, each Fab encoded may be identical exceptfor the amino acid sequence contained within the variable loops of thecomplementarity determining regions (CDRs) of the Fab fragment. In analternative or additional embodiment, amino acid sequences within theindividual V_(H) and/or V_(L) regions may differ as well.

Display libraries may be expressed in a number of possible hostsincluding, but not limited to yeast, bacteriophage, bacteria andretroviruses. Additional display technologies that may be used includeribosome-display, microbead-display and protein-DNA linkage techniques.In a preferred embodiment, Fab display libraries are expressed in yeastor in bacteriophages (also referred to herein as “phages” or “phageparticles”. When expressed, the Fabs decorate the surface of the phageor yeast where they can interact with a given antigen. An antigen thatincludes a glycan or other antigen from a desired target may be used toselect phage particles or yeast cells expressing antibody fragments withthe highest affinity for that antigen. The DNA sequence encoding the CDRof the bound antibody fragment can then be determined through sequencingusing the bound particle or cell. In one embodiment, positive selectionis used in the development of antibodies. In some embodiments, negativeselection is utilized in the development of antibodies. In someembodiments, both positive and negative selection methods are utilizedduring multiple rounds of selection in the development of antibodiesusing display libraries.

In yeast display, cDNA encoding different antibody fragments areintroduced into yeast cells where they are expressed and the antibodyfragments are “displayed” on the cell surface as described by Chao etal. (Chao, G. et al., Isolating and engineering human antibodies usingyeast surface display. Nat Protoc. 2006;1(2):755-68). In yeast surfacedisplay, expressed antibody fragments may contain an additional domainthat includes the yeast agglutinin protein, Aga2p. This domain allowsthe antibody fragment fusion protein to attach to the outer surface ofthe yeast cell through the formation of disulphide bonds withsurface-expressed Aga1p. The result is a yeast cell, coated in aparticular antibody fragment. Display libraries of cDNA encoding theseantibody fragments are utilized initially in which the antibodyfragments each have a unique sequence. These fusion proteins areexpressed on the cell surface of millions of yeast cells where they caninteract with a desired antigenic target antigen, incubated with thecells. Target antigens may be covalently or otherwise modified with achemical or magnetic group to allow for efficient cell sorting aftersuccessful binding with a suitable antibody fragment takes place.Recovery may be by way of magnetic-activated cell sorting (MACS),fluorescence-activated cell sorting (FACS) or other cell sorting methodsknown in the art. Once a subpopulation of yeast cells is selected, thecorresponding plasmids may be analyzed to determine the CDR sequence.

Bacteriophage display technology typically utilizes filamentous phageincluding, but not limited to fd, F1 and M13 virions. Such strains arenon-lytic, allowing for continued propagation of the host and increasedviral titres. Examples of phage display methods that can be used to makethe antibodies of the present invention include those disclosed inMiersch et al. (Miersch, S. et al., Synthetic antibodies: Concepts,potential and practical considerations. Methods. 2012 Aug;57(4):486-98), Bradbury et al. (Bradbury, A.R. et al., Beyond naturalantibodies: the power of in vitro display technologies. Nat Biotechnol.2011 Mar;29(3):245-54), Brinkman et al. (Brinkmann, U. et al., Phagedisplay of disulfide-stabilized Fvfragments. J Immunol Methods. 1995 May11; 182(1):41-50); Ames et al. (Ames, R.S. et al., Conversion of murineFabs isolated from a combinatorial phage display library to full lengthimmunoglobulins. J Immunol Methods. 1995 Aug 18;184(2):177-86);Kettleborough et al. (Kettleborough, C.A. et al., Isolation of tumorcell-specific single-chain Fv from immunized mice using phage-antibodylibraries and the re-construction of whole antibodies from theseantibody fragments. Eur J Immunol. 1994 Apr; 24(4):952-8); Persic et al.(Persic, L. et al., An integrated vector system for the eukaryoticexpression of antibodies or their fragments after selection from phagedisplay libraries. Gene. 1997 Mar 10; 187(1):9-18); PCT application No.PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047;WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos.5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753;5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727;5,733,743 and 5,969,108, each of which is incorporated herein byreference in its entirety. Antibody fragment expression onbacteriophages may be carried out by inserting the cDNA encoding thefragment into the gene expressing a viral coat protein. The viral coatof filamentous bacteriophages is made up of five coat proteins, encodedby a single-stranded genome. Coat protein pIII is the preferred proteinfor antibody fragment expression, typically at the N-terminus. Ifantibody fragment expression compromises the function of pIII, viralfunction may be restored through coexpression of a wild type pIII,although such expression will reduce the number of antibody fragmentsexpressed on the viral coat, but may enhance access to the antibodyfragment by the target antigen. Expression of viral as well as antibodyfragment proteins may alternatively be encoded on multiple plasmids.This method may be used to reduce the overall size of infective plasmidsand enhance the transformation efficiency.

As described above, after selection of a host expressing a high affinityantibody or antibody fragment, (e.g., glycan-interacting antibodies) thecoding regions from the antibody or antibody fragment can be isolatedand used to generate whole antibodies, including human antibodies, orany other desired antigen binding fragment, and expressed in any desiredhost, including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described in detail below.

The DNA sequence encoding a high affinity antibody can be mutated foradditional rounds of selection in a process known as affinitymaturation. The term “affinity maturation”, as used herein, refers to amethod whereby antibodies are produced with increasing affinity for agiven antigen through successive rounds of mutation and selection ofantibody- or antibody fragment-encoding cDNA sequences. In some cases,this process is carried out in vitro. To accomplish this, amplificationof CDR coding sequences may be carried out using error-prone PCR toproduce millions of copies containing mutations including, but notlimited to point mutations, regional mutations, insertional mutationsand deletional mutations. As used herein, the term “point mutation”refers to a nucleic acid mutation in which one nucleotide within anucleotide sequence is changed to a different nucleotide. As usedherein, the term “regional mutation” refers to a nucleic acid mutationin which two or more consecutive nucleotides are changed to differentnucleotides. As used herein, the term “insertional mutation” refers to anucleic acid mutation in which one or more nucleotides are inserted intoa nucleotide sequence. As used herein, the term “deletional mutation”refers to a nucleic acid mutation in which one or more nucleotides areremoved from a nucleotide sequence. Insertional or deletional mutationsmay include the complete replacement of an entire codon or the change ofone codon to another by altering one or two nucleotides of the startingcodon.

Mutagenesis may be carried out on CDR-encoding cDNA sequences to createmillions of mutants with singular mutations in CDR heavy and light chainregions. In another approach, random mutations are introduced only atCDR residues most likely to improve affinity. These newly generatedmutagenic libraries can be used to repeat the process to screen forclones that encode antibody fragments with even higher affinity for thetarget antigen. Continued rounds of mutation and selection promote thesynthesis of clones with greater and greater affinity (Chao, G. et al.,Isolating and engineering human antibodies using yeast surface display.Nat Protoc. 2006;1(2):755-68).

Examples of techniques that can be used to produce antibodies andantibody fragments, such as Fabs and scFvs, include those described inU.S. Pat. Nos. 4,946,778 and 5,258, 498; Miersch et al. (Miersch, S. etal., Synthetic antibodies: Concepts, potential and practicalconsiderations. Methods. 2012 Aug;57(4):486-98), Chao et al. (Chao, G.et al., Isolating and engineering human antibodies using yeast surfacedisplay. Nat Protoc. 2006;1(2):755-68), Huston et al. (Huston, J.S. etal., Protein engineering of single-chain Fv analogs and fusion proteins.Methods Enzymol. 1991;203:46-88); Shu et al. (Shu, L. et al., Secretionof a single-gene-encoded immunoglobulin from myeloma cells. Proc NatlAcad Sci U S A. 1993 Sep 1;90(17):7995-9); and Skerra et al. (Skerra, A.et al., Assembly of a functional immunoglobulin Fvfragment inEscherichia coli. Science. 1988 May 20;240(4855):1038-41), each of whichis incorporated herein by reference in its entirety.

For some uses, including the in vivo use of antibodies (e.g.,glycan-interacting antibodies) in humans and in vitro detection assays,it may be preferable to use chimeric, humanized, or human antibodies. Achimeric antibody is a molecule in which different portions of theantibody are derived from different animal species, such as antibodieshaving a variable region derived from a murine monoclonal immunoglobulinand a human immunoglobulin constant region. Methods for producingchimeric antibodies are known in the art. (Morrison, S.L., Transfectomasprovide novel chimeric antibodies. Science. 1985 Sep20;229(4719):1202-7; Gillies, S.D. et al., High-level expression ofchimeric antibodies using adapted cDNA variable region cassettes. JImmunol Methods. 1989 Dec 20;125(1-2):191-202.; and U.S. Pat. Nos.5,807, 715; 4,816,567; and 4,816,397, which are incorporated herein byreference in their entirety).

Humanized antibodies are antibody molecules from non-human species thatbind to the desired antigen and have one or more complementaritydetermining regions (CDRs) from the nonhuman species and frameworkregions from a human immunoglobulin molecule. Often, framework residuesin the human framework regions are substituted with correspondingresidues from the CDR and framework regions of the donor antibody toalter, preferably improve, antigen binding. These frameworksubstitutions are identified by methods well known in the art, e.g., bymodeling of the interactions of the CDR and framework residues toidentify framework residues important for antigen binding, and bysequence comparison to identify unusual framework residues at particularpositions. (U.S. Pat. Nos. 5,693,762 and 5,585, 089; Riechmann, L. etal., Reshaping human antibodies for therapy. Nature. 1988 Mar24;332(6162):323-7, which are incorporated herein by reference in theirentireties). Antibodies can be humanized using a variety of techniquesknown in the art, including, for example, CDR-grafting ( EP239,400; PCTpublication WO 91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and5,585,089); veneering or resurfacing ( EP592,106; EP519,596; Padlan,E.A., A possible procedure for reducing the immunogenicity of antibodyvariable domains while preserving their ligand-binding properties. MolImmunol. 1991 Apr-May;28(4-5):489-98; Studnicka, G.M. et al.,Human-engineered monoclonal antibodies retain full specific bindingactivity by preserving non-CDR complementarity-modulating residues.Protein Eng. 1994 Jun;7(6):805-14; Roguska, M.A. et al., Humanization ofmurine monoclonal antibodies through variable domain resurfacing. ProcNatl Acad Sci U S A. 1994 Feb 1;91(3):969-73); and chain shuffling (U.S.Pat. No. 5,565,332); each of which is incorporated herein by referencein their entirety. Humanized antibodies of the present invention may bedeveloped for desired binding specificity, complement-dependentcytotoxicity, and antibody-dependent cellular-mediated cytotoxicity,etc.

In some cases, human frameworks are selected by alignment of donorantibody sequences with human framework sequences to find humanframework candidates with the highest level of homology. In some cases,framework regions may be selected from more than one human frameworkcandidate (e.g., framework regions 1-3 may be selected from onecandidate and framework region 4 may be selected from an alternativecandidate). In some cases, framework regions may be selected from humanconsensus sequences to avoid the risk of including immunogenic epitopescreated by somatic mutations. Consensus sequences are sequences formedby comparing many sequences and adopting most commonly occurringresidues at each position. In some cases, human frameworks may beselected from human germline sequences. These may be identified throughdatabase searching (e.g., using the NCBI protein database or otherdatabases).

Light and heavy chain human frameworks may be selected from the same orfrom different clones. Light and heavy chains derived from the sameclone have a greater likelihood of associating to form binding sitesthat are functional; however, the conserved nature of the interfacebetween heavy and light chains typically allows light and heavy chainsfrom different clones to associate and be functional. Frequency ofpairing between human light and heavy chain frameworks can be reviewed,for example, in Tiller et al., 2013. MAbs. 5(3): 445-70, the contents ofwhich are herein incorporated by reference in their entirety.

Residues in humanized antibody sequences may be considered for“back-mutation” to improve or restore antibody affinity lost duringhumanization. Back-mutation involves changing residues altered duringhumanization back to those present in the original non-human antibodysequence. Residues that are candidates for back-mutation may beidentified, for example, by comparison to standard conformations foundin canonical antibody structures (see Al-Lazikani, et al., 1997. J. Mol.Biol. 273: 927-48, the contents of which are herein incorporated byreference in their entirety). Unusual canonical residues may beidentified and targeted for back-mutation. In some cases, residues thatare candidates for back-mutation may be “Vernier residues”, a term usedto refer to residues in contact with CDRs. These residues have a higherlikelihood of impacting CDR positioning and conformation, and thereforantibody affinity and/or specificity (Strohl, W.R. Therapeutic AntibodyEngineering. Woodhead Publishing, Philadelphia PA. 2012. Ch. 6, p117).In some cases, human framework regions are kept constant and CDRs fromdonor antibodies are back-mutated to fit human CDR regions whilemaintaining binding through empirical methods.

Completely human antibodies (e.g., glycan-interacting antibodies) areparticularly desirable for therapeutic treatment of human patients, soas to avoid or alleviate immune reaction to foreign protein. Humanantibodies can be made by a variety of methods known in the art,including the antibody display methods described above, using antibodylibraries derived from human immunoglobulin sequences. See also, U.S.Pat. Nos. 4,444,887 and 4,716,111; and PCT publications WO98/46645,WO98/50433, WO 98/24893, WO 98/16654, WO96/34096, WO96/33735, andWO91/10741; each of which is incorporated herein by reference in itsentirety.

Human antibodies (e.g., glycan-interacting antibodies) can also beproduced using transgenic mice which are incapable of expressingfunctional endogenous immunoglobulins, but which can express humanimmunoglobulin polynucleotides. For example, the human heavy and lightchain immunoglobulin polynucleotide complexes can be introducedrandomly, or by homologous recombination, into mouse embryonic stemcells. Alternatively, the human variable region, constant region, anddiversity region may be introduced into mouse embryonic stem cells, inaddition to the human heavy and light chain polynucleotides. The mouseheavy and light chain immunoglobulin polynucleotides can be renderednonfunctional separately or simultaneously with the introduction ofhuman immunoglobulin loci by homologous recombination. In particular,homozygous deletion of the J_(H) region prevents endogenous antibodyproduction. The modified embryonic stem cells are expanded andmicroinjected into blastocysts to produce chimeric mice. The chimericmice are then bred to produce homozygous offspring which express humanantibodies. The transgenic mice are immunized in the normal fashion witha selected antigen, e.g., all or a portion of a glycan, glycoconjugateand/or polypeptide of the invention.

Thus, using such a technique, it is possible to produce useful humanIgG, IgA, IgM, IgD and IgE antibodies. For an overview of the technologyfor producing human antibodies, see Lonberg and Huszar (Lonberg, N. etal., Human antibodies from transgenic mice. Int Rev Immunol.1995;13(1):65-93). For a detailed discussion of the technology forproducing human antibodies and human monoclonal antibodies and protocolsfor producing such antibodies, see, e.g., PCT publications WO98/24893;WO92/01047; WO96/34096; WO WO96/33735; U.S. Pat. Nos. 5,413,923;5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318;5,885,793; 5,916,771; 5,939,598; 6,075,181; and 6,114,598, each of whichare incorporated by reference herein in their entirety. In addition,companies such as Abgenix, Inc. (Fremont, Calif.), Protein Design Labs,Inc. (Mountain View, Calif.) and Genpharm (San Jose, Calif.) can beengaged to provide human antibodies directed against a selected antigenusing technology similar to the above described technologies.

Once an antibody molecule of the present invention has been produced byan animal, a cell line, chemically synthesized, or recombinantlyexpressed, it can be purified (i.e., isolated) by any method known inthe art for the purification of an immunoglobulin or polypeptidemolecule, for example, by chromatography (e.g., ion exchange, affinity,particularly by affinity for the specific antigen, Protein A, and sizingcolumn chromatography), centrifugation, differential solubility, or byany other standard technique for the purification of proteins. Inaddition, the antibodies of the present invention or fragments thereofcan be fused to heterologous polypeptide sequences described herein orotherwise known in the art, to facilitate purification.

The affinity between an antibody and a target or ligand (such as anantigen used to generate a given antibody) may be measured in terms ofK_(D) using one or more binding assays as described herein. Depending onthe desired application for a given antibody, varying K_(D) values maybe desirable. High affinity antibodies typically form ligand bonds witha K_(D) of about 10⁻⁵ M or less, e.g. about 10⁻⁶ M or less, about 10⁻⁷ Mor less, about 10⁻⁸ M or less, about 10⁻⁹ M or less, about 10⁻¹⁰ M orless, about 10⁻¹¹ M or less or about 10⁻¹² M or less.

In some embodiments, antibodies of the invention may be characterizedaccording to their half maximal effective or inhibitory concentration(EC₅₀ or IC₅₀, respectively). In some cases, this value may representthe concentration of antibody necessary to inhibit cells expressing STn(e.g. kill, reduce proliferation and/or reduce one or more cellfunction) at a level equal to half of the maximum inhibition observedwith the highest concentrations of antibody. Such IC₅₀ values may befrom about 0.001 nM to about 0.01 nM, from about 0.005 nM to about 0.05nM, from about 0.01 nM to about 1 nM, from about 0.05 nM to about 5 nM,from about 0.1 nM to about 10 nM, from about 0.5 nM to about 25 nM, fromabout 1 nM to about 50 nM, from about 5 nM to about 75 nM, from about 10nM to about 100 nM, from about 25 nM to about 250 nM, from about 200 nMto about 1000 nM or more than 1000 nM.

In some embodiments, antibodies taught in the present disclosure may betested for their ability to target patient-derived cancer cells and/orcancer stem cells (CSCs). According to such embodiments, patient-derivedcancer cells may be cultured in vitro and antibodies of the presentdisclosure may be used to target such cells.

In other embodiments, patient-derived cells may be used to producepatient-derived xenograft (PDX) tumors. In some cases, pieces of primaryor metastatic solid tumors maintained as tissue structures may becollected by surgery or biopsy procedures. In some cases, fluid drainedfrom malignant ascites or pleural effusions may be used. Tumors may beimplanted as pieces or single cell suspensions, either alone or in somestudies coated with MATRIGEL® (Corning Life Sciences, Corning, NY) ormixed with human fibroblasts or mesenchymal stem cells. Sites ofimplantation may include the dorsal region of mice (subcutaneousimplantation), although implantation in the same organ as the originaltumor may be an option (orthotopic implantation, i.e. pancreas, oralcavity, ovary, mammary fat pad, brain, etc.). In addition, independentlyof the tumor origin, some approaches may include implanting primarytumors in the renal capsule in an effort to increase engraftment successrates. A variety of mouse strains having different degrees ofimmunosuppression may be used in such studies. For hormone sensitivetumors, some studies may use hormone supplementation with the intent ofincreasing engraftment rates. In some embodiments, PDX tumors may begenerated in non-obese diabetic/severe combined immunodeficiency(NOD/SCID) mice. Antibodies may be administered to mice with PDX tumorsand the effect on tumor volume may be analyzed. In some cases, PDXtumors may be dissected, subjected to cellular dissociation, and theresulting cells grown in culture. The ability of antibodies of thepresent disclosure to target these cells may be assessed in vitro.

The preparation of antibodies, whether monoclonal or polyclonal, isknown in the art. Techniques for the production of antibodies are wellknown in the art and described, e.g. in Harlow and Lane “Antibodies, ALaboratory Manual”, Cold Spring Harbor Laboratory Press, 1988 and Harlowand Lane “Using Antibodies: A Laboratory Manual” Cold Spring HarborLaboratory Press, 1999.

Targets

Glycan-interacting antibodies of the present invention may exert theireffects via binding (reversibly or irreversibly) to one or more glycanor glycan-associated or glycan-related targets. In some embodiments,glycan-interacting antibodies can be prepared from any region of thetargets taught herein. In some embodiments, targets of the presentinvention include glycans. Glycans used for generating antibodies mayinclude a chain of sugars having at least 2, at least 3, at least 4, atleast 5, at least 6, at least 7, at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, at least16, at least 17, at least 18, at least 19 or at least 20 residues. Someglycans used for generating antibodies may include from about 2 residueto about 5 residues.

In some embodiments, glycan-interacting antibody target antigens includesialic acids. N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminicacid (Neu5Gc) are the major sialic acids on mammalian cell surfaces. Ofthese, Neu5Ac is naturally produced in humans. Neu5Gc is naturallyproduced in most mammals with the exception of humans due to a mutationin the cytidine monophosphate (CMP)-N-acetylneuraminic acid hydroxylase(CMAH) gene responsible for CMP-Neu5Gc production from CMP-Neu5Ac.Neu5Gc in humans is in fact immunogenic with nearly all humansexpressing anti-Neu5Gc antibodies. Despite a lack of production, mosthuman systems include some level of Neu5Gc due to dietary intake. Theseforeign products are subsequently incorporated into human glycoproteins.Such glycoproteins are contemplated as targets of the invention. Glycantarget antigens of the present invention may include, but are notlimited to those listed in the following Table.

TABLE 1 Glycan target antigens Glycan GalNAcα-R Galα1,3Galβ1,4GlcNAcβ-RGalβ1,3GalNAcβ-R Galβ1,3GlcNAcα-R Galβ1,3GlcNAcβ1,3Galβ1,4Glcβ-RGalβ1,3GlcNAcβ-R Galβ1,4GlcNAc6Sβ-R Galβ1,4GlcNAcβ-R Galβ1,4Glcβ-RKDNα2,8Neu5Acα2,3Galβ1,4Glcβ-R KDNa2,8Neu5Gcα2,3Galβ1,4Glcβ-RNeu5,9Ac2α2,3Galβ1,3GalNAcα-R Neu5,9Ac2α2,3Galβ1,3GalNAcβ-RNeu5,9Ac2α2,3Galβ1,3GlcNAcβ-R Neu5,9Ac2α2,3Galβ1,4GlcNAcβ-RNeu5,9Ac2α2,3Galβ1,4Glcβ-R Neu5,9Ac2α2,3Galβ-R Neu5,9Ac2α2,6GalNAcα-RNeu5,9Ac2α2,6Galβ1,4GlcNAcβ-R Neu5,9Ac2α2,6Galβ1,4Glcβ-RNeu5,9Ac2α2,6Galβ-R Neu5Acα2,3Galβ1,3GalNAcα-RNeu5Acα2,3Galβ1,3GalNAcβ-R Neu5Acα2,3Galβ1,3GlcNAcβ1,3Galβ1,4Glcβ-RNeu5Acα2,3Galβ1,3GlcNAcβ-R Neu5Acα2,3Galβ1,4(Fucα1,3)GlcNAc6Sβ-RNeu5Acα2,3Galβ1,4(Fucα1,3)GlcNAcβ-R Neu5Acα2,3Galβ1,4GlcNAc6Sβ-RNeu5Acα2,3Galβ1,4GlcNAcβ-R Neu5Acα2,3Galβ1,4Glcβ-R Neu5Acα2,3Galβ-RNeu5Acα2,6(KDNα2,3)Galβ1,4Glcβ-R Neu5Acα2,6(Neu5Acα2,3)Galβ1,4Glcβ-RNeu5Acα2,6(Neu5Gcα2,3)Galβ1,4Glcβ-R Neu5Acα2,6GalNAcα-RNeu5Acα2,6Galβ1,4GlcNAcβ-R Neu5Acα2,6Galβ1,4Glcβ-R Neu5Acα2,6Galβ-RNeu5Acα2,8KDNα2,6Galβ1,4Glcβ-R Neu5Acα2,8Neu5Acα2,3Galβ1,4Glcβ-RNeu5Acα2,8Neu5Acα2,3Galβ1,4Glcβ-R Neu5Acα2,8Neu5Acα2,6Galβ1,4Glcβ-RNeu5Acα2,8Neu5Acα2,8Neu5Acα2,3Galβ1,4Glcβ-RNeu5Acα2,8Neu5Acα2,8Neu5Acα2,3Galβ1,4Glcβ-RNeu5Acα2,8Neu5Gcα2,3Galβ1,4Glcβ-R Neu5Acα2,8Neu5Gcα2,6Galβ1,4Glcβ-RNeu5Gc9Acα2,3Galβ1,4Glcβ-R Neu5Gc9Acα2,6Galβ1,4Glcβ-RNeu5Gc9Acα2,3Galβ1,3GalNAcα-R Neu5Gc9Acα2,3Galβ1,3GalNAcβ-RNeu5Gc9Acα2,3Galβ1,3GlcNAcβ-R Neu5Gc9Acα2,3Galβ1,4GlcNAcβ-RNeu5Gc9Acα2,3Galβ-R Neu5Gc9Acα2,6GalNAcα-R Neu5Gc9Acα2,6Galβ1,4GlcNAcβ-RNeu5Gc9Acα2,6Galβ-R Neu5GcOMeα2,8Neu5Acα2,3Galβ1,4Glcβ-RNeu5Gcα2,3Galβ1,3GalNAcα-R Neu5Gcα2,3Galβ1,3GalNAcβ-RNeu5Gcα2,3Galβ1,3GlcNAcβ1,3Galβ1,4Glcβ-R Neu5Gcα2,3Galβ1,3GlcNAcβ-RNeu5Gcα2,3Galβ1,4(Fucα1,3)GlcNAc6Sβ-RNeu5Gcα2,3Galβ1,4(Fucα1,3)GlcNAcβ-R Neu5Gcα2,3Galβ1,4GlcNAc6Sβ-RNeu5Gcα2,3Galβ1,4GlcNAcβ-R Neu5Gcα2,3Galβ1,4Glcβ-R Neu5Gcα2,3Galβ-RNeu5Gcα2,6GalNAcα-R Neu5Gcα2,6Galβ1,4GlcNAcβ-R Neu5Gcα2,6Galβ1,4Glcβ-RNeu5Gcα2,6Galβ-R Neu5Gcα2,8Neu5Acα2,3Galβ1,4Glcβ-RNeu5Gcα2,8Neu5Gcα2,3Galβ1,4Glcβ-R

The following abbreviations are used herein: Glc — glucose, Gal -galactose, GlcNAc - N-acetylglucosamine, GalNAc - N-acetylgalactosamine,GlcNAc6S — 6-Sulfo-N-acetylglucosamine, KDN —2-keto-3-deoxy-D-glycero-D-galactonononic acid, Neu5,9Ac2-N-acetyl-9-0-acetylneuraminic acid, Fuc — fucose and Neu5GcOMe —2-O-methyl-N-glycolylneuraminic acid. O-glycosidic bonds are presentbetween each residue in the glycans listed with α and β indicating therelative stoichiometry between the two residues joined by the bond,wherein α indicates an axial orientation and β indicates an equatorialorientation. The numbers following α and/or β, in the format x,x,indicate the carbon number of each of the carbons from each of theadjoined residues that participate in bond formation. While the glycanslisted in the previous Table represent individual glycan target antigenscontemplated, the present invention also includes embodiments whereinthe above presented glycans include different combinations of α andβ-oriented O-glycosidic bonds than the ones presented. Also in theprevious Table, R represents an entity that the glycan may be coupledwith. In some embodiments, R is a protein wherein the glycan is linkedtypically to a serine or threonine residue. In some embodiments, R is alinker molecule used to join the glycan to a substrate, such as in aglycan array. In some embodiments, R may be a linker with the formula of—(CH₂)₂CH₂NH₂ or —(CH₂)₃NHCOCH₂(OCH₂CH₂)₆NH₂. In some embodiments, R maybe biotin, albumin, ProNH₂, —CH—, —OH, —OCH₃, —OCH₂CH₃, —H, hydrido,hydroxy, alkoxyl, oxygen, carbon, sulfur, nitrogen, polyacrylamide,phosphorus, NH₂, ProNH₂═O(CH₂)₂CH₂NH₂, (OCH₂CH₂)₆NH₂,O(CH₂)₃NHCOCH₂(OCH₂CH₂)₆NH₂, the fluorescent labels 2-aminobenzamide(AB) and/or 2-aminobenzoid acid (AA), 2-aminobenzamide analog thatcontains an alkyl amine (AEAB), aminooxy- groups, methylaminooxygroups,hydrazide groups, amino lipid1,2-dihexadecyl-sn-glycero-3-phosphoethanolamine (DHPE), aminooxy (AO)functionalized DHPE and glycosylphosphatidylinositol (GPI). Withoutintending to limit the source or nature of R, this may includestructures that affect the physical spacing of glycan residue. In someembodiments, the R group may include a combination of the R groupspresented here, e.g. a biotinylated polyacrylamide. In some embodiments,the R group in combination with underlying substrates effect glycanresidue spacing.

Glycan targets of the present invention may include one or more regionsof antibody recognition. As used herein, the term “region of antibodyrecognition” refers to a segment located on any part of the molecule, anattached group or located on a region of interaction between the glycanand another molecule, including, but not limited to another glycan,protein, membrane, cell surface structure, or extracellular matrixcomponent. In some embodiments, regions of antibody recognition arelocated at interchain target sites, wherein the term “interchain” meanswithin the present polymeric chain. Interchain target sites may includeregions of antibody recognition having 1, 2, 3, 4, 5, 6, 7, 8, 9 or atleast 10 residues, bonds between residues or combinations of residuesand bonds. In some embodiments, regions of antibody recognition arelocated at regions of interaction between one or more glycan chains.Such regions may be between 2, 3, 4 or at least 5 glycan chains.

In some embodiments, regions of antibody recognition are located atregions of interaction between glycan branch chains connected to acommon parent chain. In some embodiments, regions of antibodyrecognition are located at regions of interaction between a glycanbranch chain and a parent chain. In some embodiments, regions ofantibody recognition are located at regions of interaction betweenglycans and proteins. Such regions of interaction may include chemicalbonds between the glycan and the protein, including, but not limited tocovalent bonds, ionic bonds, hydrostatic bonds, hydrophobic bonds andhydrogen bonds. In some embodiments, regions of antibody recognition arelocated at regions of interaction between glycans and other biomoleculesincluding, but not limited to lipids and nucleic acids. Such regions ofinteraction may include chemical bonds between the glycan and thebiomolecule, including, but not limited to covalent bonds, ionic bonds,hydrostatic bonds, hydrophobic bonds and hydrogen bonds.

In some embodiments, glycan targets of the present invention arecomponents of glycoconjugates. As used herein, the term “glycoconjugate”refers to any entity joined with a glycan moiety. In some embodiments,glycoconjugates are glycolipids. As used herein, the term “glycolipid”refers to a class of lipids wherein a carbohydrate moiety is covalentlyattached. In some embodiments, carbohydrate moieties present onglycolipids may be glycans. In some embodiments, lipid components ofglycolipids include ceramide moieties. Examples of glycolipidscontemplated as targets of the present invention include, but are notlimited to glyceroglycolipids (including, but not limited togalactolipids and sulfolipids), glycosphingolipids (including, but notlimited to cerebrosides (e.g., galactocerebrosides, glucocerebrosidesand sulfatides), gangliosides, globosides and glycophosphosphingolipids)and glycosylphosphatidylinositols. When located within cell membranes,glycan moieties of glycolipids are located on the extracellular side ofthe membrane where they may interact with other cells as well as cellsignaling ligands (Maccioni, H.J. et al., Organization of the synthesisof glycolipid oligosaccharides in the Golgi complex. FEBS Lett. 2011 Jun6;585(11):1691-8).

In some embodiments, glycoconjugate targets of the present invention areglycoprotein and/or proteoglycans. Glycoproteins refer to any proteinsthat are covalently bonded with glycans. Proteoglycans are a class ofproteins that are heavily glycosylated with glycans that often carry anegative charge. This property makes them very hydrophilic and importantcomponents of connective tissue.

Cancer-Related Targets

In some embodiments, targets of the present invention are cancer-relatedantigens or epitopes. As used herein, the term “cancer-related” is usedto describe entities that may be in some way associated with cancer,cancerous cells and/or cancerous tissues. Many cancer-related antigensor epitopes that include glycans have been identified that are expressedin correlation with tumor cells (Heimburg-Molinaro, J. et al., Cancervaccines and carbohydrate epitopes. Vaccine. 2011 Nov 8;29(48):8802-26).These are referred to herein as “tumor-associated carbohydrate antigens”or “TACAs.” TACAs include, but are not limited to mucin-related antigens[including, but not limited to Tn, Sialyl Tn (STn) andThomsen-Friedenreich antigen], blood group Lewis related antigens[including, but not limited to Lewis^(Y) (Le^(Y)), Lewis^(X) (Le^(X)),Sialyl Lewis^(X) (SLe^(X)) and Sialyl Lewis^(A) (SLe^(A))],glycosphingolipid-related antigens [including, but not limited to GloboH, stage-specific embryonic antigen-3 (SSEA-3) and glycosphingolipidsthat include sialic acid], ganglioside-related antigens [including, butnot limited to gangliosides GD2, GD3, GM2, fucosyl GM1 and Neu5GcGM3]and polysialic acid-related antigens. Many of such antigens aredescribed in International Publication No. WO2015054600, the contents ofwhich are herein incorporated by reference in their entirety.

In some embodiments, TACA targets of the present invention include Lewisblood group antigens. Lewis blood group antigens include a fucoseresidue linked to GlcNAc by an α1-3 linkage or an α1-4 linkage. They maybe found on both glycolipids and glycoproteins. Lewis blood groupantigens may be found in the body fluid of individuals that aresecretors of these antigens. Their appearance on red cells is due toabsorption of Lewis antigens from the serum by the red cells.

In some embodiments, TACA targets of the present invention includeLe^(Y). Le^(Y) (also known as CD174) is made up of Galβ1,4GlcNAC havingα1,2- as well as α1,3-linked fucose residues yielding theFucα(1,2)Galβ(1,4)Fucα(1,3)GlcNAc epitope. It is synthesized from the Hantigen by α1,3 fucosyltransferases which attach the α1,3 fucose to theGlcNAc residue of the parent chain. Le^(Y) may be expressed in a varietyof cancers including, but not limited to ovarian, breast, prostate,colon, lung and epithelial. Due to its low expression level in normaltissues and elevated expression level in many cancers, the Le^(Y)antigen is an attractive target for therapeutic antibodies.

In some embodiments, TACA targets of the present invention includeLe^(X). Le^(X) includes the epitope Galβ1-4(Fucα1-3)GlcNAcβ-R. It isalso known as CD15 and stage-specific embryonic antigen-1 (SSEA-1). Thisantigen was first recognized as being immunoreactive with sera takenfrom a mouse subjected to immunization with F9 teratocarcinoma cells.Le^(x) was also found to correlate with embryonic development atspecific stages. It is also expressed in a variety of tissues both inthe presence and absence of cancer, but can also be found in breast andovarian cancers where it is only expressed by cancerous cells.

In some embodiments, TACA targets of the present invention includeSLe^(A) and/or SLe^(X). SLe^(A) and SLe^(X) are made up of structuresNeu5Acα2-3Ga1β1-3(Fuca1-4)G1cNAcβ-R andNeu5Acα2-3Galβ1-4(Fucα1-3)GlcNAcβ-R, respectively. Their expression isupregulated in cancer cells. The presence of these antigens in serumcorrelates with malignancy and poor prognosis. SLe^(X) is mostly foundas a mucin terminal epitope. It is expressed in a number of differentcancers including breast, ovarian, melanoma, colon, liver, lung andprostate. In some embodiments of the present invention, SLe^(A) andSLe^(x) targets include Neu5Gc (referred to herein as GcSLe^(A) andGcSLe^(X), respectively).

In some cases, cancer-related targets of the invention may includemucins. Ishida et al demonstrate that interaction of MUC2 with dendriticcells (with anti-tumor activity) leads to dendritic cell apoptosis(Ishida, A. et al., 2008. Proteomics. 8: 3342-9, the contents of whichare herein incorporated by reference in their entirety). In someaspects, the present invention provided anti-mucin antibodies to preventdendritic cell apoptosis and support anti-tumor activity.

In some embodiments, TACA targets of the present invention includeglycolipids and/or epitopes present on glycolipids, including, but notlimited to glycosphingolipids. Glycosphingolipids include the lipidceramide linked to a glycan by the ceramide hydroxyl group. On the cellmembrane, glycosphingolipids form clusters referred to as “lipid rafts”.

In some embodiments, TACA targets of the present invention include GloboH. Globo H is a cancer-related glycosphingolipid first identified inbreast cancer cells. The glycan portion of Globo H includesFucα(1-2)Galβ(1-3)GalNAcβ(1-3)Galα(1-4)Galβ(1-4)Glcβ(1). Although foundin a number of normal epithelial tissues, Globo H has been identified inassociation with many tumor tissues including, but not limited to, smallcell lung, breast, prostate, lung, pancreatic, gastric, ovarian andendometrial tumors.

In some embodiments, cancer-related glycosphingolipid targets of thepresent invention include gangliosides. Gangliosides areglycosphingolipids having one or more sialic acid. According toganglioside nomenclature, G is used as an abbreviation for ganglioside.This abbreviation is followed by the letters M, D or T referring to thenumber of sialic acid residues attached (1, 2 or 3 respectively).Finally the numbers 1, 2 or 3 are used to refer to the order of thedistance each migrates when analyzed by thin layer chromatography(wherein 3 travels the greatest distance, followed by 2, and then 1).Gangliosides are known to be involved in cancer-related growth andmetastasis and may be expressed on the cell surface of tumor cells.Gangliosides expressed on tumor cells may include, but are not limitedto GD2, GD3, GM2 and fucosyl GM1 (also referred to herein as Fuc-GM1).In some embodiments of the present invention, glycan-interactingantibodies are directed toward GD3. GD3 is a regulator of cell growth.In some embodiments, GD3-directed antibodies are used to modulate cellgrowth and/or angiogenesis. In some embodiments, GD3-directed antibodiesare used to modulate cell attachment. GD3 associated with some tumorcells may include 9-O-acetylated sialic acid residues (Mukherjee, K. etal., 2008. J Cell Biochem. 105: 724-34 and Mukherjee, K. et al., 2009.Biol Chem. 390: 325-35, the contents of each of which are hereinincorporated by reference in their entirety). In some cases, antibodiesof the invention are selective for 9-O-acetylated sialic acid residues.Some antibodies may be specific for 9-O-acetylated GD3s. Such antibodiesmay be used to target tumor cells expressing 9-O-acetylated GD3. In someembodiments of the present invention, glycan interacting antibodies aredirected toward GM2. In some embodiments, GM2-directed antibodies areused to modulate cell to cell contact. In some embodiments, gangliosidetargets of the present invention include Neu5Gc. In some embodiments,such targets may include a GM3 variant having Neu5Gc (referred to hereinas GcGM3). The glycan component of GcGM3 is Neu5Gcα2-3Galβ1-4Glc. GcGM3is a known component of tumor cells (Casadesus, A.V. et al., 2013.Glycoconj J. 30(7):687-99, the contents of which are herein incorporatedby reference in their entirety).

In some embodiments, TACAs of the present disclosure include at leastone Neu5Gc residue.

Recombinant Antibodies

Recombinant antibodies (e.g., glycan-interacting antibodies) of theinvention may be generated using standard techniques known in the art.In some embodiments, recombinant antibodies may be anti-glycanantibodies. Further antibodies may be anti-STn antibodies (e.g.anti-GcSTn or anti-AcSTn antibodies). Recombinant antibodies of theinvention may be produced using variable domains obtained from hybridomacell-derived antibodies produced according to methods described herein.Heavy and light chain variable region cDNA sequences of antibodies maybe determined using standard biochemical techniques. Total RNA may beextracted from antibody-producing hybridoma cells and converted to cDNAby reverse transcriptase (RT) polymerase chain reaction (PCR). PCRamplification may be carried out on resulting cDNA to amplify variableregion genes. Such amplification may include the use of primers specificfor amplification of heavy and light chain sequences. In otherembodiments, recombinant antibodies may be produced using variabledomains obtained from other sources. This includes the use of variabledomains selected from one or more antibody fragment library, such as anscFv library used in antigen panning. Resulting PCR products may then besubcloned into plasmids for sequence analysis. Once sequenced, antibodycoding sequences may be placed into expression vectors. Forhumanization, coding sequences for human heavy and light chain constantdomains may be used to substitute for homologous murine sequences. Theresulting constructs may then be transfected into mammalian cells forlarge scale translation.

Anti-Tn Antibodies

In some embodiments, recombinant antibodies of the invention (e.g.,glycan-interacting antibodies) may be anti-Tn antibodies. Suchantibodies may bind to targets having Tn. Anti-Tn antibodies may bespecific for Tn or may bind other modified forms of Tn, such as Tnlinked to other moieties, including, but not limited to additionalcarbohydrate residues. In some cases anti-Tn antibodies may beanti-sialyl-Tn antibodies. Such antibodies may bind to sialylated Tnthat includes Neu5Ac and/or sialylated Tn that include Neu5Gc. Someanti-Tn antibodies may bind specifically to clusters of Tn antigen.

Anti-STn Antibodies

In some embodiments, antibodies of the invention (e.g.,glycan-interacting antibodies) may specifically bind to STn. Anti-STnantibodies of the invention may be categorized by their binding tospecific portions of STn antigens and/or by their specificity for AcSTnversus GcSTn. In some cases, anti-STn antibodies of the invention areGroup 1 antibodies. “Group 1” antibodies according to the invention areantibodies capable of binding AcSTn and GcSTn. Such antibodies may alsobe referred to herein as pan-STn antibodies due to their ability toassociate with a wider range of STn structures. In some embodiments,Group 1 antibodies may associate with the portion of STn indicated bythe large oval in FIG. 1A. In some cases, anti-STn antibodies of theinvention are Group 2 antibodies. “Group 2” antibodies, accoding to theinvention, are antibodies capable of binding STn as well as some relatedstructures that include an O-linkage to serine or threonine. In someembodiments, Group 2 antibodies may associate with glycans that includea sialylated galactose residue. In some cases, Group 2 antibodies mayassociate with the portion of STn indicated by the large oval in FIG.1B. Some Group 2 antibodies preferably bind to structures with AcSTnover structures with GcSTn. Further anti-STn antibodies may be Group 3antibodies. As referred to herein, “Group 3” antibodies are antibodiescapable of binding STn, but may also bind a broader set of relatedstructures. Unlike Group 2 antibodies, Group 3 antibodies do not requirethat such structures have an O-linkage to serine or threonine. In someembodiments, Group 3 antibodies may associate with the portion of STnindicated by the large oval in FIG. 1C. Finally, some anti-STnantibodies of the invention may be Group 4 antibodies. As referred toherein, “Group 4” antibodies are capable of binding to both AcSTn andGcSTn as well as the un-sialylated Tn antigen, and therefore havebroader specificity. In some embodiments, Group 4 antibodies mayassociate with the portion of STn indicated by the large oval in FIG.1D.

In some cases, anti-STn antibodies of the invention may bindspecifically to clusters of STn on a particular antigen or cell surface.Some such antibodies may recognize epitopes formed by the clustering ofSTn, including epitopes that include areas of contact betweenneighboring STn structures. Such epitopes may be formed by theclustering of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more STn structures.

In some embodiments, anti-STn antibodies of the present disclosure maybe used bind cellular proteins carrying STn. Such antibodies may beuseful for targeting cellular proteins associated with cancer cells thatare distinguishable from similar proteins in non-cancerous cells by STnexpression. In some cases, such proteins may include cell surfaceproteins. Cancer cell surface proteins carrying STn may be targeted byanti-STn antibodies during cancer treatment and/or diagnosis. Cellsurface proteins carrying STn may be identified using mass spectrometryand/or using immunological methods (e.g., FACS analysis,immunoprecipitation, immunoblotting, ELISA, etc.). In some cases,cellular proteins carrying STn may include cancer cell markers, cancerstem cell markers, and/or cancer stem cell signaling proteins. In someembodiments, cellular proteins carrying STn may include, but are notlimited to CD44, CD133, CD117, integrins, Notch, and Hedgehog.

Antibody Components

In some cases, antibodies or antigen binding fragments thereof of theinvention may include variable domain and/or CDR amino acid sequencesprovided herein. Some antibodies or antigen binding fragments mayinclude different combinations of such sequences. In some cases,antibodies or antigen binding fragments of the invention may include oneor more of the variable domain sequences listed in the following Table.Residues indicated with an “X” may be absent or selected from any aminoacid residues. Light chain variable domains presented in the Table maybe expressed with or without a C-terminal arginine residue. This residuetypically links light chain variable domains with light chain constantdomains and may be expressed as part of the light chain constant domaininstead of the light chain variable domain. In some cases, antibodies orantigen binding fragments thereof may include an amino acid sequencewith from about 50% to about 99.9% sequence identity (e.g. from about50% to about 60%, from about 55% to about 65%, from about 60% to about70%, from about 65% to about 75%, from about 70% to about 80%, fromabout 75% to about 85%, from about 80% to about 90%, from about 85% toabout 95%, from about 90% to about 99.9%, from about 95% to about 99.9%,about 97%, about 97.5%, about 98%, about 98.5%, about 99%, about 99.5%,about 99.6%, about 99.7% or about 99.8%) with one or more of thevariable domain sequences listed in the following Table. In some cases,antibodies or antigen binding fragments thereof of the invention mayinclude an amino acid sequence having one or more fragments of any ofthe sequences listed in the following Table.

TABLE 2 Variable domain sequences Antibody ID Number Variable domainSequence SEQ ID NO 7D3-2C10 Heavy chainQVQLLQYDAELVKPGGSVKISCKASGYTFTDHAIHWVKQKPEQGLEWIGYFSPGNDDIKYSEKFKGKATLTADKSSSTAYMQLNSLTSEDSAVYFCKRSITTPYWGQGTLVTV SA 1 7D3-2C10 Light chainDIQMNQSPSSLSASLGDTITITCHASQNINVWLSWYQQKPGNIPKLLIYKVSNLHTGVPSRFSGSGSGTGFTLTISSLQ PEDIATYYCQQDQSYPYTFGGGTKLKK 2A5-2G12 Heavy chain QVQLQQSDAELVKPGASVKISCKASGYTFTDHAIHWVKQKPEQGLEWIGYISPGNDDIKYNEKFKGKATLTADKSSSTAYMQLNSLTSEDSAVYFCKRSITTSYWGQGTLVTV SA 3 A5-2G12 Light chainNIVMTQSPKSMSMSVGERVTLTCKASENVVIYVSWYQQKPEQSPKLLIYGASNRYTGVPDRFTGSGSATDFTLTISS VQAEDLADYHCGQGYSYPYTFGGGTKLEIKR4 1A5-2C9 Heavy chain QVQLQQSDAELVKPGASVKISCKASGYTFTDHAIHWVKQKPEQGLEWIGYVSPGNGDIKYNEKFKGKATLTADKSSSTAYMQLNSLTSEDSAVYFCKRSLIGDYWGQGTTLT VSS 5 1A5-2C9 Light chainDIVMTQSQKFMSSSVGDRVTITCKASQNVGTAVAWYQQKPGQSPKFLIYSASNRYTGVPDRFTGSGSGTDFTLTIS NMQSEDLADYFCQQYSSYRLTFGGGTKLEIK6 4D9-2C11 Heavy chain QVQLQQSDAELVKPGASVKISCKASGYTFTDHAIHWVKQKPEQGLEWIGYLSPGNDDIKYSEKFKDKATLTADKSSSTAYMQLNSLTSEDSAVYFCKRSIGGDHWGQGTTLTV SS 7 4D9-2C11 Light chainDIQMNQSPSSLSASLGDTITITC14ASQNINVWLNWYQQKPGNIPKLLIYKASNLHTGVPSRFSGSGSGTGFTLTIGSL QPEDIATYYCQQGQSYPFTFGGGTKLEIKR8 2F4-1E2 Heavy chain QVQLQQSDAELVKPGASVKISCKASGYTFTDHAIHWVKQKPEQGLEWIGYISPGNGDIKYNEKFKGKATLTADKSSSTAYMQLNSLTSEDSAVYFCQRQLGQGYWGQGTTLT VSS 9 2F4-1E2 Light chainDVVMTQTPLSLPVSLGDQASISCRSSQSLVHSYGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQNTHVPYTFGGGTKLEIKR 10 2F4-1H8 Heavy chainQVQLQQSDAELVKPGASVKISCKASGYTFTDHAIHWVKQKPEQGLEWIGYISPGNGDIKYNEKFKGKATLTADKSSSTAYMQLNSLTSEDSAVYFCQRQLGQGYWGQGTTLT VSS 9 2F4-1H8 Light chainDVVMTQTPLSLPVSLGDQASISCRSSQSLVHSYGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQNTHVPYTFGGGTKLEIKR 10 2C6-2F11 Heavy chainQVQLQQSDAELGKPGASVKISCKASGYTFSDHAIHWVKQKPEQGLEWIGYISPGNDDIKYNEKFKGKATLTADKSSSTAYMQLNSLTSEDSAVYFCERSMIGVYWGQGTLVT VSA 11 2C6-2F11 Light chainDVVMTQTPLSLTVSLGDQASISCRFSQSLVQSNGNTYLQWYLQKPGQSPKLLIYKVSNRFCGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHAPLTFGAGTKLELK 12 2B2-2A7 Heavy chainQVQLQQSDAELVKPGASVKISCKTSGYTFTDHAIHWVKQKPEQGLEWIGYISPGNGDIKYNEKFKGKATLTADKSSSTAYMQLSSLTPEDSAVYFCKISYYGIWGQGTTLTVSS 13 2B2-2A7 Light chainDIQMTQSPASLSVSVGESVTITCRLSEDIYSNLAWFQQRPGKSPQLLVYKATNLADGVPSRFSGSGSGTQYSLKINSL QSEDFGTYYCQHFWGTPFTFGSGTKVEIK 145G2-1B3 Heavy chain QVQLQQSDAELVKPGASVKISCKASGYTFTDHAIHWVKQKPEQGLEWIGYFSPGNDDIKYNEKFKVKATLTADKSSSTAYMQLTSLTSEDSAVYFCKRSYYGDWGQGTTLTV SS 15 5G2-1B3 Light chainDIQMTQSPASLSVSVGETVTITCRASENIYSHLAWYQQKQGKSPQLLVYGATNLADGVPSRFSGSGSGTQFSLKIH SLQSEDFGSYYCQHFWGAPFTFGSGTKLEIK16 7A6-2A2 Heavy chain QIQLQQSDAELVKPGTSVKMSCKASGYTFTDHAIHWVKQKPEQGLEWIGYFSPGNDDIKYNVKFKGKATLTADKSSSTAYMQLNSLTSEDSAVYFCSVGYALDYWGLGTTL TVSS 17 7A6-2A2 Light chainNIVMTQSPKSMSMSVGERVTLTCKASENVVTYVSWYQQKPEQSPKLLIYGASNRYTGVPDRFTGSGSATDFTLTISS VQAEDLADYHCGQGYSYPYTFGGGTKLEIKR18 10C9-2G7 Heavy chain QVQLQQSDAELVKPGTTVKISCKASGYTFTDHAIHWVKEKPEQGLEWIGYISPGNDDIKYSEKFKGKATLTADKSSSTAYMQLNSLTSDDSAVYFCKRSLSTPYWGQGTLVTV SA 19 10C9-2G7 Light chain Unknown1C11-2G9 Heavy chain Unknown 1C11-2G9 Light chainDIVMTQSPSSLTVTAGEKVTMSCRSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPYTFGGGTKLEIKR 20 1F6-1B7 (also sequence of1F6-1C10) Heavy chain QVQLQQSDAELVKPGASVKISCKASGYTFTDHAIHWVMQMPEQGLEWIGYISPGNGDVKYSERFKGRATLTADKSSSSAYMQLNSLTSEDSAVYFCKRSLSTPYWGQGTLVT VS 21 1F6-1B7 (also sequence of1F6-1C10) Light chain DIVMTQSPSSLTVTAGERVTMSCKSSQSLLNSGNQKSYLTWYQQKPGQPPKLLISWASTRDSGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQSDYSYPYTFGGGTKLEIKR 22 2G12-2B2 Heavy chainQVQLQQSDXELVKPGASVKISCKASGYTFTDHAIHWVKQKPEQGLEWIGYFSPGNDDIKYNEKFRGKATLTADKSSSTAYMQLNSLSSDDSAVYFCKRSLSTPYWGQGTLXTV SA 23 2G12-2B2 Light chainDIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNRGNHKNYLTWYRQKPGLPPKLLIYWASTRESGVPDRFTGSGSGTDFALTISSVQAEDLAVYYCQNDYTYPYTFGGGTKLEIKR 24 5E6-2E7 Heavy chainQVQLQQSDAELVKPGASMKISCKASGYTFTDHAIHWVKQKPEQGLEWIGYISPGNGDIKYNEKFKVKATLTADKSSSTAYMQLNSLTSEDSAVYFCKRSITTPYWGQGTLVTV SA 25 5E6-2E7 Light chainDIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGKTKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCKNDYSYPYTFGGGTKLEIKR 26 9E5-1A8 Heavy chainQVQLQQSDAELVKPGASVKISCKTSGYTFTDHAIHWVKQKPEQGLEWIGYISPGNDDIKYTEKFKGKVTLTADKSSSTAYMQLNSLTSEDSAVYFCKRSITTPYWGQGTLVTVSA 27 9E5-1A8 Light chain Unknown9F11-1F7 Heavy chain QVQLQQSDAELVKPGASMKISCKASGYTFTDHAIHWVKQKPEQGLEWIGYISPGNGDIKYNEKFKVKATLTADKSSSTAYMQLNSLTSEDSAVYFCKRSITTPYWGQGTLVTV SA 25 9F11-1F7 Light chainDIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGKTKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCKNDYSYPYTFGGGTKLEIKR 26 10F4-2F2 Heavy chainQVQLQQSDAELVKPGASVKISCKASGYTFTDHAIHWVKQKPEQGLEWIGYISPGNGDIKYDEKFKGKATLTADKSSSTAYMQLNSLTSEDSAVYFCKRSITTSYWGQGTLVTV SA 28 10F4-2F2 Light chainNIVMTQSPKSMSMSVGERVTLTCKASENVVTYVSWYQQKPEQSPKLLIYGASNRYTGVPDRFTGSGSATDFTLTISS VQAEDLADYHCGQGYSYPYTFGGGTKLEIKR18 2B8-2F10 Heavy chain QVQLQQSDAELVKPGASVKISCKASGYTFTDHAIHWVKQKPEQGLEWIGYISPGNDDIKYNEKFKGKATLTADKSSSTAYMQLNSLTSEDSAVFFCKRSITTSYWGQGTLVTV SA 29 2B8-2F10 Light chainUnknown 4G8-1E3 Heavy chain QVQLQQSDAELVKPGASVKISCKASGYIFTDHAIHWVKQKPEQGLEWIGYISPGNGDIKYNEKFKGKATLTADKSSSTAYMHLNSLTSEDSAVYFCKRSITTSYWGQGTLVTVS A 30 4G8-1E3 Light chainDIQMNQSPSSLSASLGDTITITCHASQHINFWLSWYQQKPGNIPKLLIYKASNLHTGVPSRFSGSGSGTGFTLTISSLLP EDVATYYCQQDQSYPYMFGGGTKLEIKR31 6B11-2E3 Heavy chain QVQLQQSDAELVKPGASVKISCKASGYTFTDHAIHWVKQKPEQGLEWIGYISPGNDDIKYNEKFKGKATLTADKSSSTAYMLLNSLTSEDSAVYFCKRSITTSYWGQGTLVTV SA 32 6B11-2E3 Light chainNIVMTQSPKSMSMSVGERVTLTCKASENVVTYVSWYQQKPEQSPKLLIYGASNRYTGVPDRFTGSGSATDFTLTISS VQAEDLADYHCGQGYSYPYTFGGGTKLEIKR18 8C2-2D6 Heavy chain QVQLQQSDAELVKPGASVKISCKASGYTFTDHAIHWVKQKPEQGLEWIGYISPGNGDIKYNEKFKGKATLTADTSSTTAYMQLNSLTSEDSAMYFCKRSITTSYWGQGTLVTV SA 33 8C2-2D6 Light chainNIVMTQSPKSMSMSVGERVTLTCKASENVVTYVSWYQQKPEQSPKLLIYGASNRYTGVPDRFTGSGSATDFTLTISS VQAEDLADYHCGQGYSYPYTFGGGTKLEIKR18 8C2-2D6 Light chain (V2) DIQMNQSPSSLSASLGDTITITCHASQNINVWLSWYQQKPGNIPKLLIYKASNLYTGVPSRFSGSGSGTGFTLTISSLQ PEDVATYYCQHDQSYPYTFGGGTKLEIK 347D4-2A2-2F2 Heavy chain QVQLQQSDAELVKPGASVKISCKASGYIFTDHAIHWVKQKPEQGLEWIGYISPGNGDIKYIEKFRGKATLTADKSSSTAYMQLNSLTSEDSAVYFCKRSLSTPYWGQGTLVTVS A 35 7D4-2A2-2F2 Light chainNILMTQSPKSMSMSVGERVTLTCKASENVVNYVSWYQQKPEQSPKLLIFGASNRYSGVPDRFTGSGSATDFTLTISSVQAEDLADYHCGSKWITSYPYTFGGGTKLEIKR 36 7D4-1H12-2B3 Heavy chainQVQLQQSDAELVKPGASVKISCKASGYIFTDHAIHWVKQKPEQGLEWIGYISPGNGDIKYIEKFRGKATLTADKSSSTAYMQLNSLTSEDSAVYFCKRSLSTPYWGQGTLVTVS A 35 7D4-1H12-2B3 Light chainNILMTQSPKSMSMSVGERVTLTCKASENVVNYVSWYQQKPEQSPKLLIYGASNRYSGVPDRFTGSGSATDFTLTISSVQAEDLADYHCGARVTSYPYTFGGGTKLEIKR 37 2C2-2C5 Heavy chainQVQLQQSDAELVKPGTSVKISCRASGYTFTDHAIHWVKQKPEQGLEWIGYISPGNGDIKYNEKFKGKATLTADKSSSTAYMQLNSLTSDDSAVYFCKRSITTPYWGQGTTLTVS S 38 2C2-2C5 Light chainSFVMTQTPKFLLVSAGDRVTITCKASQSVNNNVAWYQQKPGQSPKQLIYYASNRYTGVPDRFTGSGYGTDFTFTIY TVQAEDLAVYFCQQGYSSPWTFGGGTKLK 3910F4-2A9 Heavy chain QVQLQQSDAELVKPGASVKISCKASGYTFTDHAIHWVKQKPEQGLEWIGYISPGNGDIKYDEKFKGKATLTADKSSSTAYMQLNSLTSEDSAVYFCKRSITTSYWGQGTLVTV SA 28 3F1 Heavy chainQVQLQQSDAELVKPGASVKISCKASGYTFTDHAIHWVKQKPEQGLDWIGYISPGNGDIKYNEKFKDKVTLTADKSSSTACMHLNSLTSEDSAVYFCKRSLLALDYWGQGTTLT VSS 40 3F1 Light chainDIVMTQSHKFMSTSVGDRVSITCKASQDVGTNIAWYQQKPGRSPKVLIYSASTRHTGVPDRFTGSGSGTDFTLTIS NVQSEDLTDYFCQQYSSFPLTFGVGTKLELK41 3F1 Heavy chain (with C80S mutation)QVQLQQSDAELVKPGASVKISCKASGYTFTDHAIHWVKQKPEQGLDWIGYISPGNGDIKYNEKFKDKVTLTADKSSSTASMHLNSLTSEDSAVYFCKRSLLALDYWGQGTTLT VSS 42

In some cases, antibodies or antigen binding fragments thereof of theinvention may include one or more of the CDR amino acid sequences listedin the following Table. Residues indicated with an “X” may be absent orselected from any amino acid residues. In some cases, antibodies orantigen binding fragments thereof may include an amino acid sequencewith from about 50% to about 99.9% sequence identity (e.g. from about50% to about 60%, from about 55% to about 65%, from about 60% to about70%, from about 65% to about 75%, from about 70% to about 80%, fromabout 75% to about 85%, from about 80% to about 90%, from about 85% toabout 95%, from about 90% to about 99.9%, from about 95% to about 99.9%,about 97%, about 97.5%, about 98%, about 98.5%, about 99%, about 99.5%,about 99.6%, about 99.7% or about 99.8%) with one or more of the CDRsequences listed in the following Table. In some cases, antibodies orantigen binding fragments thereof of the invention may include an aminoacid sequence having one or more fragments of any of the sequenceslisted in the following Table.

TABLE 3 CDR sequences Antibody ID Number CDR Sequence SEQ ID NO 7A6-2A2CDR-H1 GYTFTDHAIHWV 43 2B2-2A7 CDR-H1 GYTFTDHAIHWV 43 5G2-1B3 CDR-H1GYTFTDHAIHWV 43 4D9-2C11 CDR-H1 GYTFTDHAIHWV 43 2F4-1E2 CDR-H1GYTFTDHAIHWV 43 2F4-1H8 CDR-H1 GYTFTDHAIHWV 43 1A5-2C9 CDR-H1GYTFTDHAIHWV 43 1F6-1B7 (also sequence of 1F6-1C10) CDR-H1 GYTFTDHAIHWV43 2C2-2C5 CDR-H1 GYTFTDHAIHWV 43 2G12-2B2 CDR-H1 GYTFTDHAIHWV 4310C9-2G7 CDR-H1 GYTFTDHAIHWV 43 2C6-2F11 CDR-H1 GYTFSDHAIHWV 447D4-2A2-2F2 CDR-H1 GYIFTDHAIHWV 45 7D4-1H12-2B3 CDR-H1 GYIFTDHAIHWV 457D3-2C10 CDR-H1 GYTFTDHAIHWV 43 8C2-2D6 CDR-H1 GYTFTDHAIHWV 43 9E5-1A8CDR-H1 GYTFTDHAIHWV 43 5E6-2E7 CDR-H1 GYTFTDHAIHWV 43 9F11-1F7 CDR-H1GYTFTDHAIHWV 43 4G8-1E3 CDR-H1 GYIFTDHAIHWV 45 10F4-2F2 CDR-H1GYTFTDHAIHWV 43 10F4-2A9 CDR-H1 GYTFTDHAIHWV 43 6B11-2E3 CDR-H1GYTFTDHAIHWV 43 2B8-2F10 CDR-H1 GYTFTDHAIHWV 43 7A5-2G12 CDR-H1GYTFTDHAIHWV 43 7A6-2A2 CDR-H2 FSPGNDDIKY 46 2B2-2A7 CDR-H2 ISPGNGDIKY47 5G2-1B3 CDR-H2 FSPGNDDIKY 46 4D9-2C11 CDR-H2 LSPGNDDIKY 48 2F4-1E2CDR-H2 ISPGNGDIKY 47 2F4-1H8 CDR-H2 ISPGNGDIKY 47 1A5-2C9 CDR-H2VSPGNGDIKY 49 1F6-1B7 (also sequence of 1F6-1C10) CDR-H2 ISPGNGDVKY 502C2-2C5 CDR-H2 ISPGNGDIKY 47 2G12-2B2 CDR-H2 FSPGNDDIKY 46 10C9-2G7CDR-H2 ISPGNDDIKY 51 2C6-2F11 CDR-H2 ISPGNDDIKY 51 7D4-2A2-2F2 CDR-H2ISPGNGDIKY 47 7D4-1H12-2B3 CDR-H2 ISPGNGDIKY 47 7D3-2C10 CDR-H2FSPGNDDIKY 46 8C2-2D6 CDR-H2 ISPGNGDIKY 47 9E5-1A8 CDR-H2 ISPGNDDIKY 515E6-2E7 CDR-H2 ISPGNGDIKY 47 9F11-1F7 CDR-H2 ISPGNGDIKY 47 4G8-1E3CDR-H2 ISPGNGDIKY 47 10F4-2F2 CDR-H2 ISPGNGDIKY 47 10F4-2A9 CDR-H2ISPGNGDIKY 47 6B11-2E3 CDR-H2 ISPGNDDIKY 51 2B8-2F10 CDR-H2 ISPGNDDIKY51 7A5-2G12 CDR-H2 ISPGNDDIKY 51 7A6-2A2 CDR-H3 SVGYALDY 52 2B2-2A7CDR-H3 KISYYGI 53 5G2-1B3 CDR-H3 KRSYYGD 54 4D9-2C11 CDR-H3 KRSIGGDH 552F4-1E2 CDR-H3 QRQLGQGY 56 2F4-1H8 CDR-H3 QRQLGQGY 56 1A5-2C9 CDR-H3KRSLIGDY 57 1F6-1B7 (also sequence of 1F6-1C10) CDR-H3 KRSLSTPY 582C2-2C5 CDR-H3 KRSITTPY 59 2G12-2B2 CDR-H3 KRSLSTPY 58 10C9-2G7 CDR-H3KRSLSTPY 58 2C6-2F11 CDR-H3 ERSMIGVY 60 7D4-2A2-2F2 CDR-H3 KRSLSTPY 587D4-1H12-2B3 CDR-H3 KRSLSTPY 58 7D3-2C10 CDR-H3 KRSITTPY 59 8C2-2D6CDR-H3 KRSITTSY 61 9E5-1A8 CDR-H3 KRSITTPY 59 5E6-2E7 CDR-H3 KRSITTPY 599F11-1F7 CDR-H3 KRSITTPY 59 4G8-1E3 CDR-H3 KRSITTSY 61 10F4-2F2 CDR-H3KRSITTSY 61 10F4-2A9 CDR-H3 KRSITTSY 61 6B11-2E3 CDR-H3 KRSITTSY 612B8-2F10 CDR-H3 KRSITTSY 61 7A5-2G12 CDR-H3 KRSITTSY 61 7A6-2A2 CDR-L1ENVVTY 62 2B2-2A7 CDR-L1 EDIYSN 63 5G2-1B3 CDR-L1 ENIYSH 64 4D9-2C11CDR-L1 QNINVW 65 2F4-1E2 CDR-L1 QSLVHSYGNTY 66 2F4-1H8 CDR-L1QSLVHSYGNTY 66 1A5-2C9 CDR-L1 QNVGTA 67 1F6-1B7 (also sequence of1F6-1C10) CDR-L1 QSLLNSGNQKSY 68 2C2-2C5 CDR-L1 QSVNNN 69 2G12-2B2CDR-L1 QSLLNRGNHKNY 70 2C6-2F11 CDR-L1 QSLVQSNGNTY 71 7D4-2A2-2F2 CDR-L1ENVVNY 72 7D4-1H12-2B3 CDR-L1 ENVVNY 72 7D3-2C10 CDR-L1 QNINVW 658C2-2D6 CDR-L1 ENVVTY 62 5E6-2E7 CDR-L1 QSLLNSGKTKNY 73 9F11-1F7 CDR-L1QSLLNSGKTKNY 73 4G8-1E3 CDR-L1 QHINFW 74 10F4-2F2 CDR-L1 ENVVTY 6210F4-2A9 CDR-L1 ENVVTY 62 6B11-2E3 CDR-L1 ENVVTY 62 7A5-2G12 CDR-L1ENVVIY 75 1C11-2G9 CDR-L1 QSLLNSGNQKNY 76 7A6-2A2 CDR-L2 GASNRYT 772B2-2A7 CDR-L2 KATNLAD 78 5G2-1B3 CDR-L2 GATNLAD 79 4D9-2C11 CDR-L2KASNLHT 80 2F4-1E2 CDR-L2 KVSNRFS 81 2F4-1H8 CDR-L2 KVSNRFS 81 1A5-2C9CDR-L2 SASNRYT 82 1F6-1B7 (also sequence of 1F6-1C10) CDR-L2 WASTRDS 832C2-2C5 CDR-L2 YASNRYT 84 2G12-2B2 CDR-L2 WASTRES 85 2C6-2F11 CDR-L2KVSNRFC 86 7D4-2A2-2F2 CDR-L2 GASNRYS 87 7D4-1H12-2B3 CDR-L2 GASNRYS 877D3-2C10 CDR-L2 KVSNLHT 88 8C2-2D6 CDR-L2 GASNRYT 77 5E6-2E7 CDR-L2WASTRES 85 9F11-1F7 CDR-L2 WASTRES 85 4G8-1E3 CDR-L2 KASNLHT 80 10F4-2F2CDR-L2 GASNRYT 77 10F4-2A9 CDR-L2 GASNRYT 77 6B11-2E3 CDR-L2 GASNRYT 777A5-2G12 CDR-L2 GASNRYT 77 1C11-2G9 CDR-L2 WASTRES 85 7A6-2A2 CDR-L3GQGYSYPYT 89 2B2-2A7 CDR-L3 QHFWGTPFT 90 5G2-1B3 CDR-L3 QHFWGAPFT 914D9-2C11 CDR-L3 QQGQSYPFT 92 2F4-1E2 CDR-L3 SQNTHVPYT 93 2F4-1H8 CDR-L3SQNTHVPYT 93 1A5-2C9 CDR-L3 QQYSSYRLT 94 1F6-1B7 (also sequence of1F6-1C10) CDR-L3 QSDYSYPYT 95 2C2-2C5 CDR-L3 QQGYSSPWT 96 2G12-2B2CDR-L3 QNDYTYPYT 97 2C6-2F11 CDR-L3 SQSTHAPLT 98 7D4-2A2-2F2 CDR-L3GSKWITSYPYT 99 7D4-1H12-2B3 CDR-L3 GARVTSYPYT 100 7D3-2C10 CDR-L3QQDQSYPYT 101 8C2-2D6 CDR-L3 GQGYSYPYT 89 5E6-2E7 CDR-L3 KNDYSYPYT 1029F11-1F7 CDR-L3 KNDYSYPYT 102 4G8-1E3 CDR-L3 QQDQSYPYM 103 10F4-2F2CDR-L3 GQGYSYPYT 89 10F4-2A9 CDR-L3 GQGYSYPYT 89 6B11-2E3 CDR-L3GQGYSYPYT 89 7A5-2G12 CDR-L3 GQGYSYPYT 89 1C11-2G9 CDR-L3 QNDYSYPYT 104

In some cases, antibodies of the present disclosure may include heavychain variable domains having one or more CDR amino acid sequences fromthe CDR sequence groups listed in the following Table. Residuesindicated with an “X” may be absent or selected from any amino acidresidues. In some cases, antibodies or antigen binding fragments thereofmay include an amino acid sequence with from about 50% to about 99.9%sequence identity (e.g. from about 50% to about 60%, from about 55% toabout 65%, from about 60% to about 70%, from about 65% to about 75%,from about 70% to about 80%, from about 75% to about 85%, from about 80%to about 90%, from about 85% to about 95%, from about 90% to about99.9%, from about 95% to about 99.9%, about 97%, about 97.5%, about 98%,about 98.5%, about 99%, about 99.5%, about 99.6%, about 99.7% or about99.8%) with one or more of the CDR sequences listed in the followingTable. In some cases, antibodies may include an amino acid sequencehaving one or more fragments of any of the sequences listed in thefollowing Table.

TABLE 4 VH CDR sequence groups Clone ID CDR-H1 SEQ ID NO CDR-H2 SEQ IDNO CDR-H3 SEQ ID NO 8C2-2D6 GYTFTDHAIH 105 YISPGNGDIKYNEKFKG 107 SITTSY114 4G8-1E3 GYIFTDHAIH 106 YISPGNGDIKYNEKFKG 107 SITTSY 114 2G12-2B2GYTFTDHAIH 105 YFSPGNDDIKYNEKFRG 108 SLSTPY 115 5G2-1B3 GYTFTDHAIH 105YFSPGNDDIKYNEKFKV 109 SYYGD 116 5E6-2E7 GYTFTDHAIH 105 YISPGNGDIKYNEKFKV110 SITTPY 117 2C2-2C5 GYTFTDHAIH 105 YISPGNGDIKYNEKFKG 107 SITTPY 1179F11-1F7 GYTFTDHAIH 105 YISPGNGDIKYNEKFKV 110 SITTPY 117 1F6-1C10GYTFTDHAIH 105 YISPGNGDVKYSERFKG 137 SLSTPY 115 7D3-2C10 GYTFTDHAIH 105YFSPGNDDIKYSEKFKG 138 SITTPY 117 7A5-2G12 GYTFTDHAIH 105YISPGNDDIKYNEKFKG 113 SITTSY 114 10F4-2A9 GYTFTDHAIH 105YISPGNGDIKYDEKFKG 139 SITTSY 114 2F4-1E2 GYTFTDHAIH 105YISPGNGDIKYNEKFKG 107 QLGQGY 140 2C6-2F11 GYTFSDHAIH 136YISPGNDDIKYNEKFKG 113 SMIGVY 141 6B11-2E3 GYTFTDHAIH 105YISPGNDDIKYNEKFKG 113 SITTSY 114 3F1 GYTFTDHAIH 105 YISPGNGDIKYNEKFKD111 SLLALDY 118 CC49 GYTFTDHAIH 105 YFSPGNDDFKYNEKFKG 112 SLNMAY 119B72.3 GYTFTDHAIH 105 YISPGNDDIKYNEKFKG 113 SYYGH 120 ConsensusGYTFTDHAIH 105 YISPGNGDIKYNEKFKG 107 SITTSY 114

In some cases, antibodies of the present disclosure may include lightchain variable domains having one or more CDR amino acid sequences fromthe CDR sequence groups listed in the following Table. Residuesindicated with an “X” may be absent or selected from any amino acidresidues. In some cases, antibodies or antigen binding fragments thereofmay include an amino acid sequence with from about 50% to about 99.9%sequence identity (e.g. from about 50% to about 60%, from about 55% toabout 65%, from about 60% to about 70%, from about 65% to about 75%,from about 70% to about 80%, from about 75% to about 85%, from about 80%to about 90%, from about 85% to about 95%, from about 90% to about99.9%, from about 95% to about 99.9%, about 97%, about 97.5%, about 98%,about 98.5%, about 99%, about 99.5%, about 99.6%, about 99.7% or about99.8%) with one or more of the CDR sequences listed in the followingTable. In some cases, antibodies may include an amino acid sequencehaving one or more fragments of any of the sequences listed in thefollowing Table.

TABLE 5 VL CDR sequence groups Clone ID CDR-L1 SEQ ID NO CDR-L2 SEQ IDNO CDR-L3 SEQ ID NO 8C2-2D6 KASENVVTYVS 121 GASNRYT 77 GQGYSYPYT 898C2-2D6(V2) HASQNINVWLS 142 KASNLYT 147 QHDQSYPTY 148 4G8-1E3HASQHINFWLS 122 KASNLHT 80 QQDQSYPYM 103 2G12-2B2 KSSQSLLNRGNHKNYLT 123WASTRES 85 QNDYTYPYT 97 5G2-1B3 RASENIYSHLA 124 GATNLAD 79 QHFWGAPFT 915E6-2E7 KSSQSLLNSGKTKNYLT 125 WASTRES 85 KNDYSYPYT 102 2C2-2C5KASQSVNNNVA 126 YASNRYT 84 QQGYSSPWT 96 1F6-1C10 KSSQSLLNSGNQKSYLT 143WASTRDS 83 QSDYSYPYT 95 7D3-2C10 HASQNINVWLS 142 KVSNLHT 88 QQDQSYPYT101 7A5-2G12 KASENVVIYVS 144 GASNRYT 77 GQGYSYPYT 89 10F4-2A9KASENVVTYVS 121 GASNRYT 77 GQGYSYPYT 89 2F4-1E2 RSSQSLVHSYGNTYLH 145KVSNRFS 81 SQNTHVPYT 93 2C6-2F11 RFSQSLVQSNGNTYLQ 146 KVSNRFC 86SQSTHAPLT 98 6B11-2E3 KASENVVTYVS 121 GASNRYT 77 GQGYSYPYT 89 3F1KASQDVGTNIA 127 SASTRHT 130 QQYSSFPLT 133 CC49 KSSQSLLYSGNQKNYLA 128WASARES 131 QQYYSYPLT 134 B72.3 RASENIYSNLA 129 AATNLAD 132 QHFWGTPYT135

In some cases, antibodies or antigen binding fragments of the inventionmay be encoded by a nucleotide sequence that includes one or more of thevariable domain sequences listed in the following Table. Residueslabeled “N” may be absent or selected from nucleotides A, C, G or T. Insome cases, antibodies or antigen binding fragments thereof may beencoded by a nucleotide sequence that includes a sequence with fromabout 50% to about 99.9% sequence identity (e.g. from about 50% to about60%, from about 55% to about 65%, from about 60% to about 70%, fromabout 65% to about 75%, from about 70% to about 80%, from about 75% toabout 85%, from about 80% to about 90%, from about 85% to about 95%,from about 90% to about 99.9%, from about 95% to about 99.9%, about 97%,about 97.5%, about 98%, about 98.5%, about 99%, about 99.5%, about99.6%, about 99.7% or about 99.8%) with one or more of the variabledomain sequences listed in the following Table. In some cases,antibodies or antigen binding fragments thereof of the invention may beencoded by a nucleotide sequence that includes one or more fragments ofany of the sequences listed in the following Table.

TABLE 6 Variable domain nucleotide sequences Antibody ID Number Variablechain Sequence SEQ ID NO 7D3-2C10 Heavy chainCAGGTTCAGTTGCTGCAGTATGACGCTGAGTTGGTG AAACCTGGGGGGTCAGTGAAGATATCGTGCAAGGCCTCTGGCTACACCTTCACTGACCATGCTATTCACTGGGTGAAGCAGAAGCCTGAACAGGGCCTGGAATGGAT TGGATATTTTTCTCCCGGAAATGATGATATTAAGTA149 CAGTGAGAAGTTCAAGGGCAAGGCCACACTGACTGCAGACAAGTCCTCCAGCACTGCCTACATGCAGCTCAACAGCCTGACATCTGAGGATTCTGCAGTGTATTTCTGTAAAAGATCCATTACTACGCCTTACTGGGGCCAAG GGACTCTGGTCACTGTCTCTGCA 7D3-2C10Light chain GACATCCAGATGAACCAGTCTCCATCCAGTCTGTCTGCATCCCTTGGAGACACAATTACCATCACTTGCCATGCCAGTCAGAACATTAATGTTTGGTTAAGCTGGTACCAGCAGAAACCAGGAAATATTCCTAAACTATTGATCTATAAGGTTTCCAACTTGCACACAGGCGTCCCATCAAGGTTTAGTGGCAGTGGATCTGGAACAGGTTTCACATTAACCATCAGCAGCCTGCAGCCTGAAGACATTGCCACTTACTACTGTCAACAGGATCAAAGTTATCCGTAC ACGTTCGGAGGGGGGACCAAGCTGAAAAAAA 1507A5-2G12 Heavy chain CAGGTTCAGCTGCAGCAGTCTGACGCTGAGTTGGTGAAACCTGGGGCTTCAGTGAAGATATCCTGCAAGGCCTCTGGCTACACCTTCACTGACCATGCTATTCACTGG GTGAAGCAGAAGCCTGAACAGGGCCTGGAATGGATTGGATATATTTCTCCCGGAAATGATGATATTAAGTA CAATGAGAAGTTCAAGGGCAAGGCCACACTGACTGCAGACAAATCCTCCAGCACTGCCTACATGCAGCTCAACAGCCTGACATCTGAGGATTCTGCAGTGTATTTCTGTAAAAGATCCATTACTACGTCTTACTGGGGCCAAG GGACTCTGGTCACTGTCTCTGCA 1517A5-2G12 Light chain AACATTGTAATGACCCAATCTCCCAAATCCATGTCCATGTCAGTAGGAGAGAGGGTCACCTTGACCTGCAA GGCCAGTGAGAATGTGGTTATTTATGTTTCCTGGTATCAACAGAAACCAGAGCAGTCTCCTAAACTGCTGATATACGGGGCATCCAACCGGTACACTGGGGTCCCCGATCGCTTCACAGGCAGTGGATCTGCAACAGATTTCACTCTGACCATCAGCAGTGTGCAGGCTGAAGACCTTGCAGATTATCACTGTGGACAGGGTTACAGCTATCCGTA CACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACG 152 1A5-2C9 Heavy chain CAGGTTCAGTTGCAGCAGTCTGACGCTGAGTTGGTGAAACCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGCTACACCTTCACTGACCATGCCATTCATTGG GTGAAGCAGAAGCCTGAACAGGGCCTGGAATGGATTGGATATGTTTCTCCCGGAAATGGTGATATTAAGTA CAATGAGAAGTTCAAGGGCAAGGCCACACTGACTGCAGACAAATCCTCCAGCACTGCCTACATGCAGCTCAACAGCCTGACATCGGAGGATTCTGCAGTGTATTTCTGTAAAAGATCTTTAATTGGAGACTATTGGGGCCAAG GCACCACTCTCACAGTCTCCTCA 153 1A5-2C9Light chain GACATTGTGATGACCCAGTCTCAAAAATTCATGTCCTCATCAGTAGGAGACAGGGTCACCATCACCTGCAAGGCCAGTCAGAATGTGGGTACTGCTGTAGCCTGGTATCAACAGAAACCAGGACAATCTCCTAAATTTCTGATTTACTCGGCATCCAATCGGTACACTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACGATCAGCAATATGCAGTCTGAAGACCTGGCA 154GATTATTTCTGCCAGCAATATAGCAGCTATCGTCTG ACGTTCGGTGGAGGCACCAAGCTGGAAATCAAAC4D9-2C11 Heavy chain CAGGTTCAGCTGCAGCAGTCTGACGCTGAATTGGTGAAACCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGCTACACCTTCACTGACCATGCTATTCACTGG GTGAAGCAGAAGCCTGAACAGGGCCTGGAATGGATTGGATATCTTTCTCCCGGAAATGATGATATTAAGTA CAGTGAGAAGTTCAAGGACAAGGCCACACTGACTGCAGACAAATCCTCCAGCACTGCCTACATGCAGCTCAACAGCCTGACATCTGAGGATTCTGCAGTGTATTTCT GTAAAAGATCCATAGGGGGGGACCACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA 155 4D9-2C11 Light chainGACATCCAGATGAACCAGTCTCCATCCAGTCTGTCTGCATCCCTTGGAGACACAATTACCATCACTTGCCATGCCAGTCAGAACATTAATGTTTGGTTAAACTGGTACCAGCAGAAACCAGGAAATATTCCTAAACTATTGATCTATAAGGCTTCCAACTTGCACACAGGCGTCCCATCAAGGTTTAGTGGCAGTGGATCTGGAACAGGTTTCACATTAACCATCGGCAGCCTGCAGCCTGAAGACATTGCCACTTACTACTGTCAACAGGGTCAAAGTTATCCGTTC ACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACG156 2F4-1E2 Heavy chain CAGGTTCAGCTGCAGCAGTCTGACGCTGAGTTGGTGAAACCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGCTACACCTTCACTGACCATGCTATTCACTGG GTGAAACAGAAGCCTGAACAGGGCCTGGAATGGATTGGATATATTTCTCCCGGAAATGGTGATATTAAGTA TAATGAGAAGTTCAAGGGCAAGGCCACACTGACTGCAGACAAATCCTCCAGCACTGCCTACATGCAGCTCAACAGCCTGACATCTGAGGATTCTGCAGTGTATTTCT GTCAAAGACAACTGGGACAAGGCTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA 157 2F4-1E2 Light chainGATGTTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTGTACACAGTTATGGAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATTTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCA GGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAATACACATGTTCCGTACACGTTCGGAGGGGGGACCAA GCTGGAAATAAAACG 158 2F4-1H8 Heavychain CAGGTTCAGCTGCAGCAGTCTGACGCTGAGTTGGTGAAACCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGCTACACCTTCACTGACCATGCTATTCACTGG GTGAAACAGAAGCCTGAACAGGGCCTGGAATGGATTGGATATATTTCTCCCGGAAATGGTGATATTAAGTA TAATGAGAAGTTCAAGGGCAAGGCCACACTGACTGCAGACAAATCCTCCAGCACTGCCTACATGCAGCTCAACAGCCTGACATCTGAGGATTCTGCAGTGTATTTCT GTCAAAGACAACTGGGACAAGGCTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA 157 2F4-1H8 Light chainGATGTTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTGTACACAGTTATGGAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATTTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCA GGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAATACACATGTTCCGTACACGTTCGGAGGGGGGACCAA GCTGGAAATAAAACG 158 2C6-2F11 Heavychain CAGGTTCAGCTGCAGCAGTCTGACGCTGAGTTGGGGAAACCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGCTACACCTTCAGTGACCATGCTATTCACTGG GTGAAGCAGAAGCCTGAACAGGGCCTGGAATGGATTGGATATATCTCTCCCGGAAACGATGATATTAAGTA CAATGAGAAGTTCAAGGGCAAGGCCACACTGACTGCAGACAAATCCTCCAGCACTGCCTACATGCAGCTCAACAGCCTGACATCTGAGGATTCTGCAGTGTATTTCTGTGAAAGATCGATGATTGGGGTTTACTGGGGCCAAG GGACTCTGGTCACTGTCTCTGCA 1592C6-2F11 Light chain GATGTTGTGATGACCCAAACTCCACTCTCCCTGACTGTCAGTCTTGGCGATCAAGCCTCCATCTCTTGCAGATTTAGTCAGAGCCTTGTACAAAGTAATGGAAATACCTATTTACAGTGGTATCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATTTACAAAGTCTCCAACCGATTTTGTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCA GGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAGTACACATGCTCCGCTCACGTTCGGTGCTGGGACCAA GCTGGAGCTGAAAC 160 2B2-2A7 Heavychain CAGGTTCAGCTGCAGCAGTCTGACGCTGAGTTGGTGAAACCTGGGGCTTCAGTGAAGATATCCTGCAAGACTTCTGGCTACACCTTCACTGACCATGCAATTCACTGG GTGAAGCAGAAGCCTGAACAGGGCCTGGAATGGATTGGATATATTTCTCCCGGAAATGGTGATATTAAGTA CAATGAGAAGTTCAAGGGCAAGGCCACCCTGACTGCAGACAAATCCTCCAGCACTGCCTATATGCAGCTCAGCAGCCTGACACCTGAGGATTCTGCAGTGTATTTCTGTAAAATATCTTACTACGGTATTTGGGGCCAAGGCA CCACTCTCACAGTCTCCTCA 161 2B2-2A7Light chain GACATCCAGATGACTCAGTCTCCAGCCTCCCTATCTGTATCTGTGGGAGAGTCTGTCACCATCACATGTCGACTAAGTGAAGATATTTACAGTAATTTAGCATGGTTTCAGCAGAGACCGGGAAAATCTCCTCAGCTCCTGGTTTATAAAGCAACAAACTTAGCAGACGGTGTGCCATCAAGGTTCAGTGGCAGTGGATCAGGCACACAGTATTCCCTCAAGATCAACAGCCTGCAGTCTGAAGATTTTGGGACTTATTACTGTCAACATTTTTGGGGTACTCCATTCA CGTTCGGCTCGGGGACCAAGGTGGAAATAAAAC162 5G2-1B3 Heavy chain CAGGTTCAGCTGCAGCAGTCTGACGCTGAGTTGGTGAAACCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGCTACACCTTCACTGACCATGCTATTCACTGG 163GTGAAGCAGAAGCCTGAACAGGGCCTGGAATGGAT TGGATATTTTTCTCCCGGAAATGATGATATTAAGTATAATGAGAAGTTCAAGGTCAAGGCCACACTGACTGCAGACAAATCCTCCAGCACTGCCTACATGCAACTCACCAGCCTGACATCTGAAGATTCTGCAGTGTATTTCTGTAAAAGATCTTACTACGGTGATTGGGGCCAAGGCAC CACTCTCACAGTCTCCTCA 5G2-1B3 Lightchain GACATCCAGATGACTCAGTCTCCAGCCTCCCTATCTGTTTCTGTGGGAGAAACTGTCACCATCACATGTCGAGCAAGTGAGAATATTTACAGTCATTTAGCATGGTATCAACAGAAACAGGGAAAATCTCCTCAACTCCTGGTCTATGGTGCAACTAACTTAGCAGATGGTGTGCCATCAAGGTTCAGTGGCAGTGGATCAGGCACACAGTTTTCCCTCAAGATCCACAGCCTGCAGTCTGAAGATTTTGGGAGTTATTACTGTCAACATTTTTGGGGTGCTCCATTCA CGTTCGGCTCGGGGACAAAGTTGGAAATAAAAC164 7A6-2A2 Heavy chain CAAATTCAGCTGCAGCAGTCTGACGCTGAGTTGGTGAAACCTGGGACTTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACCTTCACTGACCATGCTATTCACTGG GTGAAGCAGAAGCCTGAACAGGGCCTGGAATGGATTGGATATTTTTCTCCCGGAAATGATGATATTAAGTATAATGTGAAGTTCAAGGGCAAGGCCACACTGACTGCAGACAAATCCTCCAGCACTGCCTACATGCAGCTCAACAGCCTGACATCTGAAGATTCTGCAGTGTATTTCTGTTCGGTGGGATACGCCCTTGACTACTGGGGCCTAGG CACCACTCTCACAGTCTCCTCA 165 7A6-2A2Light chain AACATTGTAATGACCCAATCTCCCAAATCCATGTCCATGTCAGTAGGAGAGAGGGTCACCTTGACCTGCAA GGCCAGTGAGAATGTGGTTACTTATGTTTCCTGGTATCAACAGAAACCAGAGCAGTCTCCTAAACTGCTGATATACGGGGCATCCAACCGGTACACTGGGGTCCCCGATCGCTTCACAGGCAGTGGATCTGCAACAGATTTCACTCTGACCATCAGCAGTGTGCAGGCTGAAGACCTTGCAGATTATCACTGTGGACAGGGTTACAGCTATCCGTA CACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACG 166 10C9-2G7 Heavy chain CAGGTTCAGCTGCAACAGTCTGACGCTGAGTTGGTGAAACCTGGGACTACAGTGAAGATATCCTGCAAGGCTTCTGGCTACACTTTCACTGACCATGCTATTCACTGGGTGAAGGAGAAGCCTGAACAGGGCCTGGAATGGATC GGATATATTTCTCCCGGAAATGATGATATTAAGTACAGTGAGAAGTTCAAGGGCAAGGCCACACTGACTGC AGACAAATCCTCCAGCACTGCTTACATGCAGCTCAACAGCCTGACATCTGATGATTCTGCAGTGTATTTCTGTAAAAGATCGCTTAGTACGCCTTACTGGGGCCAAGGG ACTCTGGTCACTGTCTCTGCA 167 10C9-2G7Light chain TTTTTAATACGACTCCCTATAGGGCAAGCAGTGGTATCAATGCAGATTACAAGGGGGAAAGGCATCAGACC AGCATGGGCATCAAGGTGGAATCACAGACTCTGGTCTTCATATCCATACTGTTTGGGTTATATGGAGCTGATGGGAACACATTAATGACCCAATCTCCCACATCCATGTACATGTCAGTAGGAGAGAGGGTCACCTTGACTTGCA 168AGGCCAGTGAGAATGAGATTAATTATGTTTCCTGGTATCAACAGAAACCAGAGCAGTCTCCTAAACTGTTGATATACGGGGCATCCAACCGGTACTCTGGGGTCCCCGATCGCTTCACAGGCAGTGGATCTGCAACAGATTTCACTCTGACCATCAGCAGTGTGCAGGCTGAAGACCTTGCAGATTATCCCTGTGGAGCAAGGGATTAACTAGCTA TCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGGGC 1C11-2G9 Heavy chain Unknown 1C11-2G9 Light chainGACATTGTGATGACACAGTCTCCATCCTCCCTGACT GTGACAGCAGGAGAGAAGGTCACTATGAGCTGCAGGTCCAGTCAGAGTCTGTTAAACAGTGGAAATCAAAA GAACTACTTGACCTGGTACCAGCAGAAACCAGGGCAGCCTCCTAAACTGTTGATCTACTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGAACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGTCAGAATGATTATAGTTATCCGTACACGTTCGGAGGGGGGA CCAAGCTGGAAATAAAACG 169 1F6-1B7(also sequence of 1F6-1C10) Heavy chainCAGGTTCAGCTGCAGCAGTCTGACGCTGAGTTGGTGAAACCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGCTACACCTTCACTGACCATGCTATTCACTGGGTGATGCAGATGCCTGAACAGGGCCTGGAATGGATTGGATATATTTCTCCCGGAAATGGTGATGTTAAGTAC AGTGAGAGGTTCAAGGGCAGGGCCACACTGACTGCAGACAAATCCTCCAGCTCTGCCTACATGCAGCTCAACAGCCTGACATCTGAGGATTCTGCAGTTTATTTCTGTAAAAGATCGCTTAGTACGCCTTACTGGGGCCAAGGG ACTCTGGTCACTGTCTCTG 170 1F6-1B7(also sequence of 1F6-1C10) Light chainGACATTGTGATGACACAGTCTCCATCCTCCCTGACT GTGACAGCAGGAGAGAGGGTCACTATGAGCTGCAAGTCCAGTCAGAGTCTGTTAAACAGTGGAAATCAAAA GAGCTACTTGACCTGGTACCAGCAGAAACCAGGGCAGCCTCCTAAACTGTTGATCTCCTGGGCATCCACTAGGGATTCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGAACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGTCAGAGTGATTATAGTTATCCGTACACGTTCGGAGGGGGGA CCAAGCTGGAAATAAAACG 171 2G12-2B2Heavy chain CAGGTTCAGCTGCAGCAGTCTGACGNTGAGTTGGTGAAACCGGGGGCTTCAGTGAAGATATCCTGTAAGGCTTCTGGCTACACCTTCACTGACCATGCTATTCACTGG GTGAAGCAGAAGCCTGAACAGGGCCTGGAATGGATTGGATATTTTTCTCCCGGAAATGATGATATTAAGTA CAATGAGAAGTTTAGGGGCAAGGCCACACTGACTGCAGACAAATCCTCCAGCACTGCCTACATGCAGCTCAACAGCCTGTCATCTGATGATTCTGCAGTGTATTTCTGTAAAAGATCGCTTAGTACGCCTTACTGGGGCCAAGG GACTCTGGNCACTGTCTCTGCA 172 2G12-2B2Light chain GACATTGTGATGACACAGTCTCCATCCTCCCTGACTGTGACAGCAGGAGAGAAAGTCACTATGAGCTGCAA 173GTCCAGTCAGAGTCTGTTAAACCGTGGAAATCATAAGAACTACTTGACCTGGTACCGGCAGAAACCAGGGCTGCCTCCTAAACTGTTGATTTACTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGAACAGATTTCGCTCTCACCATCAGCAGTGTTCAGGCTGAAGACCTGGCAGTTTATTACTGTCAGAATGATTATACTTATCCGTACACGTTCGGAGGGGGGAC CAAGCTGGAGATAAAACG 5E6-2E7 Heavychain CAGGTTCAGCTGCAGCAGTCTGACGCTGAGTTGGTGAAACCTGGGGCTTCAATGAAGATTTCCTGCAAGGCTTCTGGCTACACCTTCACTGACCATGCTATTCACTGG GTGAAGCAGAAGCCTGAACAGGGCCTGGAATGGATTGGATATATTTCTCCCGGAAATGGTGATATTAAGTA CAATGAGAAGTTCAAGGTCAAGGCCACACTGACTGCAGACAAATCCTCCAGCACTGCCTACATGCAGCTCAACAGCCTGACATCTGAGGATTCTGCAGTGTATTTCTGTAAAAGATCGATTACTACGCCTTACTGGGGCCAAG GGACTCTGGTCACTGTCTCTGCA 174 5E6-2E7Light chain GACATTGTGATGACACAGTCTCCATCCTCCCTGACTGTGACAGCAGGAGAGAAGGTCACTATGAGCTGCAA GTCCAGTCAGAGTCTGTTAAACAGTGGAAAAACAAAGAACTACTTGACGTGGTACCAGCAGAAACCAGGG CAGCCTCCTAAACTGTTGATCTACTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGAACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGTAAGAATGATTATAGTTATCCGTACACGTTCGGAGGGGGG ACCAAGCTGGAAATAAAACG 175 9E5-1A8Heavy chain CAGGTTCAGCTGCAGCAGTCTGACGCTGAATTGGTGAAGCCTGGGGCTTCAGTGAAGATATCCTGCAAGACTTCTGGCTACACCTTCACTGACCATGCTATTCACTGG GTGAAGCAGAAGCCTGAACAGGGCCTGGAATGGATTGGATATATCTCTCCCGGAAATGATGATATTAAGTA CACTGAGAAGTTCAAGGGCAAGGTCACACTGACTGCAGACAAATCCTCCAGCACTGCCTACATGCAGCTCAACAGCCTGACATCTGAGGATTCTGCAGTCTATTTCTGTAAAAGATCGATTACTACGCCTTACTGGGGCCAAG GGACTCTGGTCACTGTCTCTGCA 176 9E5-1A8Light chain TTTTTATACGCCACTTTCTAATACGCCTCACTATAGGGCAAGCAGTGGTATCAACGCAGATTACAAAGGGGA AAGGAATCAGACCGACTCGCGCATCAAGATGGAATCACAGACTCTGGTCTTCATATCCAGTACGCTCGGGGACTATGGAGNGGAACAGTACATTTTAATGACCCAAT GTCCCAAAGGCAAGAACATGTCAGTAGGAGAGAGGGTCACTCAGAGTGCAAGGCCAGGAGAAATCAAAAC ACTTATGTTTCCTGGTATCAACAGAAACCAGAGCANNCTNTAAAATGNNGATTACGGGGCATCCAACCGGG AATCTGGGGTCNCCGATCGCTTCACAGGCAGTGGATCTGGAACAGATTTCACTCTCACCATCAGCAGTGTGC AGGCTGAAGACCNGGCAGTNTTCACTGTGGACAGGGNTACAGTTATCCGTACACGTTCGGAGGGGGGACCA AGCTGAAAAAAACGGGC 177 9F11-1F7Heavy chain CAGGTTCAGCTGCAGCAGTCTGACGCTGAGTTGGTGAAACCTGGGGCTTCAATGAAGATTTCCTGCAAGGCTTCTGGCTACACCTTCACTGACCATGCTATTCACTGG GTGAAGCAGAAGCCTGAACAGGGCCTGGAATGGATTGGATATATTTCTCCCGGAAATGGTGATATTAAGTA CAATGAGAAGTTCAAGGTCAAGGCCACACTGACTGCAGACAAATCCTCCAGCACTGCCTACATGCAGCTCAACAGCCTGACATCTGAGGATTCTGCAGTGTATTTCTGTAAAAGATCGATTACTACGCCTTACTGGGGCCAAG GGACTCTGGTCACTGTCTCTGCA 1749F11-1F7 Light chain GACATTGTGATGACACAGTCTCCATCCTCCCTGACTGTGACAGCAGGAGAGAAGGTCACTATGAGCTGCAA GTCCAGTCAGAGTCTGTTAAACAGTGGAAAAACAAAGAACTACTTGACGTGGTACCAGCAGAAACCAGGG CAGCCTCCTAAACTGTTGATCTACTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGAACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGTAAGAATGATTATAGTTATCCGTACACGTTCGGAGGGGGG ACCAAGCTGGAAATAAAACG 175 10F4-2F2Heavy chain CAGGTTCAGCTGCAGCAGTCTGACGCTGAGTTGGTGAAACCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGCTACACCTTCACTGACCATGCTATTCACTGG GTGAAGCAGAAGCCTGAACAGGGCCTGGAATGGATTGGATATATTTCTCCCGGAAATGGTGATATTAAGTA CGATGAGAAGTTTAAGGGCAAGGCCACACTGACTGCAGACAAATCCTCCTCCACTGCCTACATGCAGCTCAACAGCCTGACATCTGAAGATTCTGCAGTGTATTTCTGTAAAAGATCGATTACTACCTCTTACTGGGGCCAAG GGACTCTGGTCACTGTCTCTGCA 17810F4-2F2 Light chain AACATTGTAATGACCCAATCTCCCAAATCCATGTCCATGTCAGTAGGAGAGAGGGTCACCTTGACCTGCAA GGCCAGTGAGAATGTGGTTACTTATGTTTCCTGGTATCAACAGAAACCAGAGCAGTCTCCTAAACTGCTGATATACGGGGCATCCAACCGGTACACTGGGGTCCCCGATCGCTTCACAGGCAGTGGATCTGCAACAGATTTCACTCTGACCATCAGCAGTGTGCAGGCTGAAGACCTTGCAGATTATCACTGTGGACAGGGTTACAGCTATCCGTA CACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACG 166 2B8-2F10 Heavy chain CAGGTTCAGCTGCAGCAGTCTGACGCTGAGTTGGTGAAACCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGCTACACCTTCACTGACCATGCTATTCACTGG GTGAAGCAGAAGCCTGAACAGGGCCTGGAATGGATTGGATATATTTCTCCCGGAAATGATGATATTAAGTA CAATGAGAAGTTCAAGGGCAAGGCCACACTGACTGCAGACAAGTCCTCCAGCACTGCCTACATGCAGCTCAACAGCCTGACATCTGAGGATTCTGCAGTGTTTTTCTGTAAAAGATCGATTACTACCTCTTACTGGGGCCAAG GGACTCTGGTCACTGTCTCTGCA 1792B8-2F10 Light chain TTNATAGGACTCAATATAGGGCAAGCAGTGGTATTAACGCCGAGTACATGGGGAGGGCAAGGGCAGAAAGT 180CACTTTCAGTGAGGATACACCATCAGCATGAGGGTCCTTGTTGAGCTCCTGGGGGGGCTGGTGTTNTGCTTTTTAGGTGTGAGATGTGACATCCAGATGAACCAGTCTCCATCCAGTCTGTNTGCATCCTTTGGAGACACAATTACCATCATTTGCCATTCCAGTCAGAACATTAATGTTTGGTTAAGATGGTACCAGCAGAAACCAGGAAATATTC CTAAAATATTGATATATAAGGGTTCCAACTTGTACACAGGCGTCCCATCAAGGTTTAGTGGCAGTGGATTTGGAACAGGTTTCACATTAACCATCAGCAGCGTGCAGCGGGAAGACATTGCCACTTACTACTGTCAACAGGATC AAAGTTATCCGTACACGTTCGGAGGGGGGACCAAGCTGAAATAAAACGGGC 4G8-1E3 Heavy chainCAGGTTCAGCTGCAGCAGTCTGACGCCGAGTTGGTGAAACCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGCTACATCTTCACTGACCATGCTATTCACTGGGTGAAGCAGAAGCCTGAACAGGGCCTGGAATGGATT GGATATATTTCTCCCGGAAATGGTGATATTAAGTACAATGAGAAGTTCAAGGGCAAGGCCACACTGACTGC AGACAAATCCTCCAGCACTGCCTACATGCATCTCAACAGCCTGACATCTGAGGATTCTGCAGTGTATTTCTGTAAAAGATCGATTACTACCTCTTACTGGGGCCAAGG GACTCTGGTCACTGTCTCTGCA 181 4G8-1E3Light chain GACATCCAGATGAACCAGTCCCCATCCAGTCTGTCTGCATCCCTTGGAGACACAATTACCATCACTTGCCATGCCAGTCAGCACATTAATTTTTGGTTAAGCTGGTACCAGCAGAAACCAGGAAATATTCCTAAACTCTTGATCTATAAGGCTTCCAACTTGCACACAGGCGTCCCATCAAGGTTTAGTGGCAGTGGATCTGGAACAGGTTTCACATTAACCATCAGCAGCCTGCTGCCTGAAGACGTTGCCACTTACTACTGTCAACAGGATCAAAGTTATCCGTAT ATGTTCGGAGGGGGGACCAAGCTGGAAATAAAACG182 6B11-2E3 Heavy chain CAGGTTCAGCTGCAGCAGTCTGACGCTGAGTTGGTGAAACCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGCTACACCTTCACTGACCATGCTATTCACTGG GTGAAGCAGAAGCCTGAACAGGGCCTGGAATGGATTGGATATATTTCTCCCGGAAATGATGATATTAAGTA CAATGAGAAGTTTAAGGGCAAGGCCACACTGACTGCAGACAAATCCTCCAGCACTGCCTACATGCTGCTCAACAGCCTGACATCTGAGGATTCTGCAGTGTATTTCTGTAAAAGATCGATTACTACCTCTTACTGGGGCCAAG GGACTCTGGTCACTGTCTCTGCA 1836B11-2E3 Light chain AACATTGTAATGACCCAATCTCCCAAATCCATGTCCATGTCAGTAGGAGAGAGGGTCACCTTGACCTGCAA GGCCAGTGAGAATGTGGTTACTTATGTTTCCTGGTATCAACAGAAACCAGAGCAGTCTCCTAAACTGCTGATATACGGGGCATCCAACCGGTACACTGGGGTCCCCGATCGCTTCACAGGCAGTGGATCTGCAACAGATTTCACTTTGACCATCAGCAGTGTGCAGGCTGAAGACCTTGCAGATTATCACTGTGGACAGGGTTACAGCTATCCGTA CACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACG 184 8C2-2D6 Heavy chain CAGGTTCAACTGCAGCAGTCTGACGCTGAGTTGGTGAAACCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGCTACACCTTCACTGACCATGCTATTCACTGG GTGAAGCAGAAGCCTGAACAGGGCCTGGAATGGATTGGATATATTTCTCCCGGAAATGGTGATATTAAGTA CAATGAGAAGTTCAAGGGTAAGGCCACACTGACTGCAGACACTTCCTCCACCACTGCCTACATGCAGCTCAACAGCCTGACATCTGAGGATTCTGCAATGTATTTCTGTAAAAGATCCATTACTACGTCTTACTGGGGCCAAG GGACTCTGGTCACTGTCTCTGCA 185 8C2-2D6Light chain AACATTGTAATGACCCAATCTCCCAAATCCATGTCCATGTCAGTAGGAGAGAGGGTCACCTTGACCTGCAA GGCCAGTGAGAATGTGGTTACTTATGTTTCCTGGTATCAACAGAAACCAGAGCAGTCTCCTAAACTGCTGATATACGGGGCATCCAACCGGTACACTGGGGTCCCCGATCGCTTCACAGGCAGTGGATCTGCAACAGATTTCACTCTGACCATCAGCAGTGTGCAGGCTGAAGACCTTGCAGATTATCACTGTGGACAGGGTTACAGCTATCCGTA CACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACG 152 8C2-2D6 Light chain (V2) GACATCCAGATGAACCAGTCTCCATCCAGTCTGTCTGCATCCCTTGGAGACACAATTACCATCACTTGCCATGCCAGTCAGAACATTAATGTTTGGTTAAGCTGGTACCAGCAGAAACCAGGAAATATTCCTAAACTATTGATCTATAAGGCTTCCAATTTGTATACAGGCGTCCCATCAAGGTTTAGTGGCAGTGGATCTGGAACAGGTTTCACATTAACCATCAGCAGCCTGCAGCCTGAAGACGTTGCCACGTACTACTGTCAACACGATCAAAGTTATCCGTAC ACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA186 7D4-2A2-2F2 Heavy chain CAGGTTCAGCTGCAGCAGTCTGACGCTGAGTTGGTGAAACCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGCTACATCTTCACTGACCATGCAATTCACTGG GTGAAGCAGAAGCCTGAACAGGGCCTGGAATGGATTGGATATATTTCTCCCGGAAATGGTGATATTAAGTA CATTGAGAAGTTCAGGGGCAAGGCCACACTGACTGCAGACAAATCCTCCAGCACTGCCTACATGCAGCTCAACAGCCTGACATCTGAGGATTCTGCAGTGTATTTCTGTAAAAGATCGCTTAGTACGCCTTACTGGGGCCAAG GGACTCTGGTCACTGTCTCTGCA 1877D4-2A2-2F2 Light chain AACATTTTAATGACCCAATCTCCCAAATCCATGTCCATGTCAGTAGGAGAGAGGGTCACCTTGACCTGCAA GGCCAGTGAGAATGTGGTTAATTATGTTTCCTGGTATCAACAGAAACCAGAGCAGTCTCCTAAACTGCTGATATTCGGGGCATCCAACCGGTACTCTGGGGTCCCCGATCGCTTCACAGGCAGTGGATCTGCAACAGATTTCACTCTGACCATCAGCAGTGTGCAGGCTGAAGACCTTGCAGATTATCACTGTGGAAGCAAGTGGATTACTAGCTA TCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACG 188 7D4-1H12-2B3 Heavy chainCAGGTTCAGCTGCAGCAGTCTGACGCTGAGTTGGTGAAACCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGCTACATCTTCACTGACCATGCAATTCACTGG 187GTGAAGCAGAAGCCTGAACAGGGCCTGGAATGGAT TGGATATATTTCTCCCGGAAATGGTGATATTAAGTACATTGAGAAGTTCAGGGGCAAGGCCACACTGACTG CAGACAAATCCTCCAGCACTGCCTACATGCAGCTCAACAGCCTGACATCTGAGGATTCTGCAGTGTATTTCTGTAAAAGATCGCTTAGTACGCCTTACTGGGGCCAAG GGACTCTGGTCACTGTCTCTGCA7D4-1H12-2B3 Light chain AACATTTTAATGACCCAATCTCCCAAATCCATGTCCATGTCAGTAGGAGAGAGGGTCACCTTGACCTGCAA GGCCAGTGAGAATGTGGTTAATTATGTTTCCTGGTATCAACAGAAACCAGAGCAGTCTCCTAAACTGCTGATATACGGGGCATCCAACCGGTACTCTGGGGTCCCCGATCGCTTCACAGGCAGTGGATCTGCAACAGATTTCACTCTGACCATCAGCAGTGTGCAGGCTGAAGACCTTGCAGATTATCACTGTGGAGCAAGGGTTACTAGCTATCC GTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACG 189 2C2-2C5 Heavy chain CAGGTTCAGCTGCAGCAGTCTGACGCTGAGTTGGTGAAACCTGGGACTTCAGTGAAGATATCCTGCAGGGCTTCTGGCTACACCTTCACTGACCATGCTATTCACTGG GTGAAGCAGAAGCCTGAACAGGGCCTGGAATGGATTGGATATATTTCTCCCGGAAATGGTGATATTAAGTA CAATGAGAAGTTCAAGGGCAAGGCCACACTGACTGCAGACAAATCCTCCAGCACTGCCTACATGCAGCTCAACAGCCTGACATCTGACGATTCTGCAGTGTATTTCTGTAAAAGATCCATTACTACGCCTTACTGGGGCCAAG GCACCACTCTCACAGTCTCCTCA 190 2C2-2C5Light chain AGTTTTGTGATGACCCAGACTCCCAAATTCCTGCTTGTGTCAGCAGGAGACAGGGTTACCATAACCTGCAA GGCCAGTCAGAGTGTGAATAATAATGTAGCTTGGTACCAACAGAAGCCAGGGCAGTCTCCTAAACAGCTGA TATACTATGCATCCAATCGCTACACTGGAGTCCCTGATCGCTTCACTGGCAGTGGATATGGGACGGATTTCACTTTCACCATCTACACTGTGCAGGCTGAAGACCTGGCAGTTTATTTCTGTCAGCAGGGTTATAGCTCTCCGTG GACGTTCGGTGGAGGCACCAAGCTGAAA 19110F4-2A9 Heavy chain CAGGTTCAGCTGCAGCAGTCTGACGCTGAGTTGGTGAAACCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGCTACACCTTCACTGACCATGCTATTCACTGG GTGAAGCAGAAGCCTGAACAGGGCCTGGAATGGATTGGATATATTTCTCCCGGAAATGGTGATATTAAGTA CGATGAGAAGTTTAAGGGCAAGGCCACACTGACTGCAGACAAATCCTCCTCCACTGCCTACATGCAGCTCAACAGCCTGACATCTGAAGATTCTGCAGTGTATTTCTGTAAAAGATCGATTACTACCTCTTACTGGGGCCAAG GGACTCTGGTCACTGTCTCTGCA 17810F4-2A9 Light chain AACATTGTAATGACCCAATCTCCCAAATCCATGTCCATGTCAGTAGGAGAGAGGGTCACCTTGACCTGCAA GGCCAGTGAGAATGTGGTTACTTATGTTTCCTGGTATCAACAGAAACCAGAGCAGTCTCCTAAACTGCTGATATACGGGGCATCCAACCGGTACACTGGGGTCCCCGATCGCTTCACAGGCAGTGGATCTGCAACAGATTTCAC 152TCTGACCATCAGCAGTGTGCAGGCTGAAGACCTTGCAGATTATCACTGTGGACAGGGTTACAGCTATCCGTA CACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACG

In some cases, antibodies or antigen binding fragments of the inventionmay include any of the IgG framework regions presented in the followingTable. In some cases, antibodies or fragments thereof may include anamino acid sequence with from about 50% to about 99.9% sequence identity(e.g. from about 50% to about 60%, from about 55% to about 65%, fromabout 60% to about 70%, from about 65% to about 75%, from about 70% toabout 80%, from about 75% to about 85%, from about 80% to about 90%,from about 85% to about 95%, from about 90% to about 99.9%, from about95% to about 99.9%, about 97%, about 97.5%, about 98%, about 98.5%,about 99%, about 99.5%, about 99.6%, about 99.7% or about 99.8%) withone or more of the constant domain sequences listed in the followingTable. In some cases, antibodies or fragments thereof of the inventionmay include an amino acid sequence having one or more fragments of anyof the sequences listed in the following Table.

TABLE 7 IgG Constant domain sequences Domain Sequence SEQ ID NO MurineIgG2a heavy chain constant domain regionsAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHH TTKSFSRTPGK 192 Murine IgG2akappa light chain constant regionRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYER HNSYTCEATHKTSTSPIVKSFNRNEC 193Human IgG1 heavy ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG 194 chain constant regionsPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK Human IgG1 lightchain constant regions RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGEC 195

In some cases, antibodies may include one or both of the amino acidsequences in the following table and/or be encoded by one or both of thenucleotide sequences presented in the following Table or optimizedversions thereof.

TABLE 8 3F1 antibody sequences Antibody Domain Sequence SEQ ID NO 3F1Heavy chain full length, amino acidsQVQLQQSDAELVKPGASVKISCKASGYTFTDHAIHWVKQKPEQGLDWIGYISPGNGDIKYNEKFKDKVTLTA DKSSSTACMHLNSLTSEDSAVYFCKRSLLALDYWGQGTTLTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHR EDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDS DGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK 196 3F1 Heavy chain full length, nucleotideATGGAGACCGACACCCTGCTGCTCTGGGTGCTGCT GCTCTGGGTGCCCGGCTCCACCGGACAGGTTCAGCTGCAGCAGTCTGACGCTGAGTTGGTGAAACCTGG GGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGCTACACCTTCACTGACCATGCTATTCACTGGGTGAAG CAAAAGCCTGAACAGGGCCTGGACTGGATTGGATATATTTCTCCCGGAAATGGTGATATTAAGTACAAT GAGAAGTTCAAGGACAAGGTCACACTGACTGCAGACAAATCCTCCAGCACTGCCTGCATGCACCTCAAC AGCCTGACATCTGAGGATTCTGCAGTGTATTTCTGCAAAAGATCCCTACTAGCTCTTGACTACTGGGGCC AAGGCACCACTCTCACAGTCTCCTCAGCTAAAACAACAGCCCCATCGGTCTATCCACTGGCCCCTGTGTG TGGAGATACAACTGGCTCCTCGGTGACTCTAGGATGCCTGGTCAAGGGTTATTTCCCTGAGCCAGTGACC TTGACCTGGAACTCTGGTTCCCTGTCCAGTGGTGT197 GCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACCCTCAGCTCAAGCGTGACTGTAACCAGCTCG ACCTGGCCCAGCCAGTCCATCACCTGCAATGTGGCCCACCCGGCAAGCAGCACCAAGGTGGACAAGAAA ATTGAGCCCAGAGGGCCCACAATCAAGCCCTGTCCTCCATGCAAATGCCCAGCACCTAACCTCTTGGGT GGACCATCCGTCTTCATCTTCCCTCCAAAGATCAAGGATGTACTCATGATCTCCCTGAGCCCCATAGTCA CATGTGTAGTCGTTGATGTGAGCGAGGATGACCCAGATGTCCAGATCAGCTGGTTTGTGAACAACGTGGA AGTGCACACTGCTCAGACACAGACGCATAGAGAGGATTACAACAGTACTCTCCGGGTTGTCAGTGCCCT CCCCATCCAGCACCAGGACTGGATGAGTGGCAAGGAGTTCAAATGCAAGGTCAACAACAAAGACCTCC CAGCGCCCATCGAGAGAACCATCTCAAAACCCAAAGGGTCAGTAAGAGCTCCACAGGTATATGTCTTGC CTCCACCAGAAGAGGAGATGACTAAGAAACAGGTCACTCTGACCTGCATGGTCACAGACTTCATGCCTG AAGACATTTACGTGGAGTGGACCAACAACGGGAAAACAGAGCTAAACTACAAGAACACTGAACCAGTC CTGGACTCTGATGGTTCTTACTTCATGTACAGCAAGCTGAGAGTGGAGAAGAAGAACTGGGTGGAGAG AAATAGCTACTCCTGTTCAGTGGTCCACGAGGGTCTGCACAATCACCACACGACTAAGAGCTTCTCCCGG ACTCCGGGTAAATAG 3F1 Light chain fulllength, amino acids DIVMTQSHKFMSTSVGDRVSITCKASQDVGTNIAWYQQKPGRSPKVLIYSASTRHTGVPDRFTGSGSGTDFTLTISNVQSEDLTDYFCQQYSSFPLTFGVGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLT KDEYERHNSYTCEATHKTSTSPIVKSFNRNEC 1983F1 Light chain full length, nucleotideATGGAGACCGACACCCTGCTGCTCTGGGTGCTGCT GCTCTGGGTGCCCGGCTCCACCGGAGACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTA GGAGACAGGGTCAGCATCACCTGCAAGGCCAGTCAGGATGTGGGCACTAATATAGCCTGGTATCAACA GAAACCAGGCCGATCTCCTAAAGTACTGATTTACTCGGCATCCACCCGGCACACTGGAGTCCCTGATCGC TTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATTAGCAATGTGCAGTCTGAAGACTTGACAG ATTATTTCTGTCAGCAATATAGCAGCTTTCCTCTCACGTTCGGTGTTGGGACCAAGCTGGAGCTGAAACG GGCAGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCA GTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAA CGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAG CACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGA CATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGTTGA 199

In some cases, antibodies or fragments thereof may include an amino acidsequence with from about 50% to about 99.9% sequence identity (e.g.,from about 50% to about 60%, from about 55% to about 65%, from about 60%to about 70%, from about 65% to about 75%, from about 70% to about 80%,from about 75% to about 85%, from about 80% to about 90%, from about 85%to about 95%, from about 90% to about 99.9%, from about 95% to about99.9%, about 97%, about 97.5%, about 98%, about 98.5%, about 99%, about99.5%, about 99.6%, about 99.7% or about 99.8%) with one or more of theamino acid sequences presented in the previous Table. In some cases,antibodies or fragments thereof may be encoded by a nucleotide sequencewith from about 50% to about 99.9% sequence identity (e.g. from about50% to about 60%, from about 55% to about 65%, from about 60% to about70%, from about 65% to about 75%, from about 70% to about 80%, fromabout 75% to about 85%, from about 80% to about 90%, from about 85% toabout 95%, from about 90% to about 99.9%, from about 95% to about 99.9%,about 97%, about 97.5%, about 98%, about 98.5%, about 99%, about 99.5%,about 99.6%, about 99.7% or about 99.8%) with one or more of thenucleotide sequences presented in the previous Table.

In some embodiments, the disclosure includes antibody fragments producedusing one or more of the antibody sequences or related variantsdescribed above. Such antibody fragments may include scFvs, Fabfragments, or any other antibody fragments, including any of thosedescribed herein.

Humanized Antibodies

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequences derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from thehypervariable region from an antibody of the recipient are replaced byresidues from the hypervariable region from an antibody of a non-humanspecies (donor antibody) such as mouse, rat, rabbit or nonhuman primatehaving the desired specificity, affinity, and capacity.

In some embodiments, fully humanized heavy and light chains may bedesigned from antibody sequences and/or with CDRs presented herein.Protein models of antibody variable regions may be generated usingexisting antibody structures as templates. Segments of starting heavyand light chain variable region amino acid sequences may be comparedwith human sequences to identify human germline antibodies with similarsequences. Series of humanized heavy and light chain variable regionsmay be designed using human variable domain framework region sequenceswith the objective that T cell epitopes be avoided. Variant humansequence segments with significant incidence of potential T cellepitopes as determined by in silico technologies may then be discarded.In some cases, some of the amino acid residues in resulting variabledomains may be mutated back to amino acids present in the original mousevariable domain. In some cases, some of the mouse residues in theresulting variable domains may be mutated to match residues present inhuman germline sequences.

Humanized heavy and light chain variable region genes may be constructedfrom overlapping oligonucleotides assembled into full length genes usingthe ligase chain reaction (LCR). LCR products may be amplified andsuitable restriction sites may be added for cloning into expressionvectors. PCR products may be cloned into intermediate vectors andconfirmed by sequencing.

For construction of expression plasmids encoding fully humanizedantibodies with human constant regions, DNA sequences encoding antibodyvariable region may be inserted into expression vectors (e.g., mammalianexpression vectors) between an upstream promoter/enhancer, for example,cytomegalovirus immediate/early promoter/enhancer (CMV IE), plus theimmunoglobulin signal sequence and a downstream immunoglobulin constantregion gene. DNA samples may then be prepared for transfection intomammalian cells.

For generation of cell lines and selection of fully humanizedantibodies, heavy and light chain plasmid DNA pairs may be transfectedinto cells for expression. In some embodiments, mammalian NS0 cells maybe used. Cell lines producing humanized antibodies may be expanded forexpression antibodies that may be harvested and purified from cellculture media.

In some embodiments, antibodies of the present disclosure may beprepared according to humanization methods known in the art. Suchmethods may include, but are not limited to CDR grafting, resurfacing,superhumanization, and human string content optimization (see, forexample, Almagro, et al., 2008. Front. Biosci. 13:1619-33). In someembodiments, empirical methods are used. Such methods may include thegeneration of large combinatorial libraries and selecting desiredvariants by enrichment technoloiges, such as phage display, yeastdisplay, ribosomal display, or other high throughput screeningtechniques. These methods may be utilized alone or in combination withframework libraries, guided selection, framework shuffling, andhumaneering.

In some embodiments, humanized antibodies may be prepared by utilizingone or more of the human variable domains presented in the followingTable. Such antibodies may include one or more of any of the CDRsequences presented herein or fragments or variants thereof that aresubstituted for the CDR sequences present in the human variable domains.In some cases, variants of the human variable domain sequences areutilized, wherein such variants have at least 50%, at least 60%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, atleast 99.5%, or at least 99.9% sequence identity to any of the humanvariable domain sequences presented in the following Table.

TABLE 9 Human variable domains Variable domain Kabat Germline SequenceSEQ ID NO VH IGHV1- CAGGTTCAGCTGGTGCAGTCTGGAGCTGAGGTG 200 18^(∗)01,AAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGC nucleotideAAGGCTTCTGGTTACACCTTTACCAGCTATGGTA TCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAGCGCTTACAATG GTAACACAAACTATGCACAGAAGCTCCAGGGCAGAGTCACCATGACCACAGACACATCCACGAGCA CAGCCTACATGGAGCTGAGGAGCCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAGA VL IGKV1-GACATCCAGATGACCCAGTCTCCATCCTCCCTGT 201 39^(∗)01,CTGCATCTGTAGGAGACAGAGTCACCATCACTT nucleotideGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAA ATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAG TGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTG CAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTC VL IGKV4-1^(∗)01, GACATCGTGATGACCCAGTCTCCAGACTCCCTGG202 nucleotide CTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTATACAGCTCCA ACAATAAGAACTACTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAGCTGCTCATTTACTG GGCATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCAC TCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAGCAATATTATAGTACT CCTCC VH IGHV1-QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGIS 203 18^(∗)01, aminoWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGR acids VTMTTDTSTSTAYMELRSLRSDDTAVYYCARVL IGKV1- DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWY 204 39^(∗)01, aminoQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFT acids LTISSLQPEDFATYYCQQSYSTP VLIGKV4-1^(∗)01, DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNK 205 amino acidsNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSG SGSGTDFTLTISSLQAEDVAVYYCQQYYSTPC

In some embodiments, humanized antibodies of the present disclosure mayinclude one or more of the human framework regions presented in thefollowing Table. Some antibodies may include framework regions with atleast 50%, at least 60%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, or at least 98% sequence identityto any of the framework regions presented in the following Table.

TABLE 10 Human framework regions Framework region, Variable domain KabatGermline Amino Acid Sequence SEQ ID NO FR1, VH IGHV1-18^(∗)01QVQLVQSGAEVKKPGASVKVSCKAS 206 FR1, VL IGKV1-39^(∗)01IQMTQSPSSLSASVGDRVTITC 207 FR1, VL IGKV4-1^(∗)01 DIVMTQSPDSLAVSLGERATINC208 FR2, VH IGHV1-18^(∗)01 WVRQAPGQGLEWMG 209 FR2, VL IGKV1-39^(∗)01WYQQKPGKAPKLLIY 210 FR2, VL IGKV4-1^(∗)01 WYQQKPGQPPKLLIY 211 FR3, VHIGHV1-18^(∗)01 RVTMTTDTSTSTAYMELRSLRSDDTAVY YCAR 212 FR3, VLIGKV1-39^(∗)01 GVPSRFSGSGSGTDFTLTISSLQPEDFATY YC 213 FR3, VLIGKV4-1^(∗)01 GVPDRFSGSGSGTDFTLTISSLQAEDVAV YYC 214 FR4, VH Humanconsensus sequence 1 WGQGTLVTVSS 215 FR4, VL Human consensus sequence 1FGQGTKVEIK 216

In some embodiments, one or more residues of humanized antibodies may beback-crossed to improve antibody binding or other properties.

In some embodiments, humanized variable domains present in antibodies ofthe present disclosure may include any of the variable domains presentedin the following Table. In some cases, antibodies include one or morevariants of these variable domains with at least 70%, at least 75%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or at least 99.5% sequence identity.

TABLE 11 Humanized variable domains mAb Chain Sequence SEQ ID NO 5G2-1B3VL0 DIQMTQSPSSLSASVGDRVTITCRASENIYSHLAWYQQKPGKAPKLLIYGATNLADGVPSRFSGSGSGTDFTLT ISSLQPEDFATYYCQHFWGAPFTFGQGTKVEIK217 5G2-1B3 VL1 DIQMTQSPSSLSASVGDRVTITCRASENIYSHLAWYQQKPGKAPKLLVYGATNLASGVPSRFSGSGSGTQFTL TISSLQPEDFATYYCQHFWGAPFTFGQGTKVEIK218 5G2-1B3 VL2 DIQMTQSPSSLSASVGDRVTITCRASENIYSHLAWYQQKPGKAPKLLVYGATNLADGVPSRFSGSGSGTQFTL TISSLQPEDFATYYCQHFWGAPFTFGQGTKVEIK219 5G2-1B3 VH0 QVQLVQSGAEVKKPGASVKVSCKASGYTFTDHAIHWVRQAPGQGLEWMGYFSPGNDDIKYNEKFKVRVT MTTDTSTSTAYMELRSLRSDDTAVYYCARSYYGDWGQGTLVTVSS 220 5G2-1B3 VH1 QVQLVQSGAEVKKPGASVKVSCKASGYTFTDHAIHWVRQAPGQGLEWMGYFSPGNDDIKYNEKFKVRVT MTADKSSSTAYMELRSLRSDDTAVYFCKRSYYGDWGQGTLVTVSS 221 5G2-1B3 VH2 QVQLVQSGAEVKKPGASVKISCKASGYTFTDHAIHWVRQAPGQGLEWIGYFSPGNDDIKYNEKFKVRATLTA DKSSSTAYMELRSLRSDDTAVYFCKRSYYGDWGQGTLVTVSS 222 5G2-1B3 VH3 EVQLVQSGAEVKKPGASVKVSCKASGYTFTDHAIHWVRQAPGQGLEWMGYFSPGNDDIKYNEKFKVRVT MTADKSSSTAYMELRSLRSDDTAVYFCKRSYYGDWGQGTLVTVSS 223 5G2-1B3 VH4 EVQLVQSGAEVKKPGASVKISCKASGYTFTDHAIHWVRQAPGQGLEWIGYFSPGNDDIKYNEKFKVRATLTA DKSSSTAYMELRSLRSDDTAVYFCKRSYYGDWGQGTLVTVSS 224 4G8-1E3 VL0 DIQMTQSPSSLSASVGDRVTITCHASQHINFWLSWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTL TISSLQPEDFATYYCQQDQSYPYMFGQGTKVEIK225 4G8-1E3 VL1 DIQMTQSPSSLSASVGDRVTITCHASQHINFWLSWYQQKPGKIPKLLIYKASNLHTGVPSRFSGSGSGTGFTL TISSLQPEDFATYYCQQDQSYPYMFGQGTKVEIK226 4G8-1E3 VL2 DIQMTQSPSSLSASVGDRITITCHASQHINFWLSWYQQKPGKIPKLLIYKASNLHTGVPSRFSGSGSGTGFTLTI SSLQPEDVATYYCQQDQSYPYMFGQGTKLEIK227 4G8-1E3 VL3 DIQMTQSPSSLSASVGDRVTITCHASQHINFWLSWYQQKPGKIPKLLIYKASNLHTGVPSRFSGSGSGTGFTL TISSLQPEDFATYYCQQDQSYPYFFGQGTKVEIK228 4G8-1E3 VL4 DIQMTQSPSSLSASVGDRITITCHASQHINFWLSWYQQKPGKIPKLLIYKASNLHTGVPSRFSGSGSGTGFTLTI SSLQPEDVATYYCQQDQSYPYFFGQGTKLEIK229 4G8-1E3 VH0 QVQLVQSGAEVKKPGASVKVSCKASGYIFTDHAIHWVRQAPGQGLEWMGYISPGNGDIKYNEKFKGRVT MTTDTSTSTAYMELRSLRSDDTAVYYCARSITTSYWGQGTLVTVSS 230 4G8-1E3 VH1 QVQLVQSGAEVKKPGASVKVSCKASGYIFTDHAIHWVRQAPGQGLEWMGYISPGNGDIKYNEKFKGRVT MTADKSSSTAYMELRSLRSDDTAVYFCKRSITTSYWGQGTLVTVSS 231 4G8-1E3 VH2 QVQLVQSGAEVKKPGASVKISCKASGYIFTDHAIHWVRQAPGQGLEWIGYISPGNGDIKYNEKFKGRATLTADKSSSTAYMHLRSLRSDDTAVYFCKRSITTSYWGQG TLVTVSS 232 4G8-1E3 VH3EVQLVQSGAEVKKPGASVKVSCKASGYIFTDHAIHW VRQAPGQGLEWMGYISPGSGDIKYNEKFKGRVTMTADKSSSTAYMELRSLRSDDTAVYFCKRSITTSYWGQ GTLVTVSS 233 4G8-1E3 VH4EVQLVQSGAEVKKPGASVKISCKASGYIFTDHAIHWVRQAPGQGLEWIGYISPGSGDIKYNEKFKGRATLTADKSSSTAYMHLRSLRSDDTAVYFCKRSITTSYWGQG TLVTVSS 234 2G12-2B2 VL0DIVMTQSPDSLAVSLGERATINCKSSQSLLNRGNHKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYTYPYTFGQGT KVEIK 235 2G12-2B2 VL2DIVMTQSPDSLAVSLGERVTMSCKSSQSLLNRGNHKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYTYPYTFGQGT KVEIK 236 2G12-2B2 VH0QVQLVQSGAEVKKPGASVKVSCKASGYTFTDHAIH WVRQAPGQGLEWMGYFSPGNDDIKYNEKFRGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARSLSTPYW GQGTLVTVSS 237 2G12-2B2 VH1QVQLVQSGAEVKKPGASVKVSCKASGYTFTDHAIH WVRQAPGQGLEWMGYFSPGNDDIKYNEKFRGRVTMTADKSSSTAYMELRSLRSDDTAVYFCKRSLSTPYW GQGTLVTVSS 238 2G12-2B2 VH2QVQLVQSGAEVKKPGASVKISCKASGYTFTDHAIHWVRQAPGQGLEWIGYFSPGNDDIKYNEKFRGRVTLTADKSSSTAYMELRSLRSDDTAVYFCKRSLSTPYWGQG TLVTVSS 239 2G12-2B2 VH3EVQLVQSGAEVKKPGASVKVSCKASGYTFTDHAIH WVRQAPGQGLEWMGYFSPGNDDIKYNEKFRGRVTMTADKSSSTAYMELRSLRSDDTAVYFCKRSLSTPYW GQGTLVTVSS 240 2G12-2B2 VH4EVQLVQSGAEVKKPGASVKISCKASGYTFTDHAIHWVRQAPGQGLEWIGYFSPGNDDIKYNEKFRGRVTLTADKSSSTAYMELRSLRSDDTAVYFCKRSLSTPYWGQG TLVTVSS 241 8C2-2D6 VL0DIQMTQSPSSLSASVGDRVTITCKASENVVTYVSWYQQKPGKAPKLLIYGASNRYTGVPSRFSGSGSGTDFTL TISSLQPEDFATYYCGQGYSYPYTFGQGTKVEIK242 8C2-2D6 VL1 NIQMTQSPSSLSASVGDRVTITCKASENVVTYVSWYQQKPGKAPKLLIYGASNRYTGVPSRFSGSGSATDFTL TISSLQPEDFATYYCGQGYSYPYTFGQGTKVEIK243 8C2-2D6 VL2 NIVMTQSPSSMSMSVGDRVTLTCKASENVVTYVSWYQQKPGKSPKLLIYGASNRYTGVPSRFSGSGSATDFTLTISSVQPEDLATYHCGQGYSYPYTFGQGTKLEIK 244 8C2-2D6(V2) VL0DIQMTQSPSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKAPKLLIYKASNLYTGVPSRFSGSGSGTDFTL TISSLQPEDFATYYCQHDQSYPYTFGQGTKVEIK245 8C2-2D6(V2) VL1 DIQMTQSPSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKIPKLLIYKASNLYTGVPSRFSGSGSGTGFTL TISSLQPEDFATYYCQHDQSYPYTFGQGTKVEIK246 8C2-2D6(V2) VL2 DIQMTQSPSSLSASVGDRITITCHASQNINVWLSWYQQKPGKIPKLLIYKASNLYTGVPSRFSGSGSGTGFTLTI SSLQPEDFATYYCQHDQSYPYTFGQGTKLEIK247 8C2-2D6(V2) VL3 DIQMNQSPSSLSASVGDRITITCHASQNINVWLSWYQQKPGKIPKLLIYKASNLYTGVPSRFSGSGSGTGFTLTI SSLQPEDFATYYCQHDQSYPYTFGQGTKLEIK248 8C2-2D6 VH0 QVQLVQSGAEVKKPGASVKVSCKASGYTFTDHAIHWVRQAPGQGLEWMGYISPGNGDIKYNEKFKGRVT MTTDTSTSTAYMELRSLRSDDTAVYYCARSITTSYWGQGTLVTVSS 249 8C2-2D6 VH1 QVQLVQSGAEVKKPGASVKVSCKASGYTFTDHAIHWVRQAPGQGLEWMGYISPGNGDIKYNEKFKGRVT MTADKSSTTAYMELRSLRSDDTAVYFCKRSITTSYWGQGTLVTVSS 250 8C2-2D6 VH2 QVQLVQSGAEVKKPGASVKISCKASGYTFTDHAIHWVRQAPGQGLEWIGYISPGNGDIKYNEKFKGRATLTADKSSTTAYMELRSLRSDDTAMYFCKRSITTSYWGQG TLVTVSS 251 8C2-2D6 VH3EVQLVQSGAEVKKPGASVKVSCKASGYTFTDHAIH WVRQAPGQGLEWMGYISPGSGDIKYNEKFKGRVTMTADKSSTTAYMELRSLRSDDTAVYFCKRSITTSYWG QGTLVTVSS 252 8C2-2D6 VH4EVQLVQSGAEVKKPGASVKISCKASGYTFTDHAIHWVRQAPGQGLEWIGYISPGSGDIKYNEKFKGRATLTADKSSTTAYMELRSLRSDDTAMYFCKRSITTSYWGQG TLVTVSS 253 3F1 VL0DIQMTQSPSSLSASVGDRVTITCKASQDVGTNIAWYQQKPGKAPKLLIYSASTRHTGVPSRFSGSGSGTDFTL TISSLQPEDFATYYCQQYSSFPLTFGQGTKVEIK254 3F1 VL1 DIQMTQSPSSLSASVGDRVTITCKASQDVGTNIAWYQQKPGKAPKVLIYSASTRHTGVPSRFSGSGSGTDFTL TISSLQPEDFATYFCQQYSSFPLTFGQGTKVEIK255 3F1 VH0 QVQLVQSGAEVKKPGASVKVSCKASGYTFTDHAIHWVRQAPGQGLEWMGYISPGNGDIKYNEKFKDRVT MTTDTSTSTAYMELRSLRSDDTAVYYCARSLLALDYWGQGTLVTVSS 256 3F1 VH1 QVQLVQSGAEVKKPGASVKVSCKASGYTFTDHAIHWVRQAPGQGLEWMGYISPGNGDIKYNEKFKDRVT MTADKSSSTAYMQLRSLRSDDTAVYFCKRSLLALDYWGQGTLVTVSS 257 3F1 VH2 QVQLVQSGAEVKKPGASVKISCKASGYTFTD14AIHWVRQAPGQGLEWIGYISPGNGDIKYNEKFKDRVTLTA DKSSSTASMHLRSLRSDDTAVYFCKRSLLALDYWGQGTLVTVSS 258 3F1 VH3 EVQLVQSGAEVKKPGASVKVSCKASGYTFTDHAIHWVRQAPGQGLEWMGYISPGSGDIKYNEKFKDRVTM TADKSSSTAYMQLRSLRSDDTAVYFCKRSLLALDYWGQGTLVTVSS 259 3F1 VH4 EVQLVQSGAEVKKPGASVKISCKASGYTFTDHAIHWVRQAPGQGLEWIGYISPGSGDIKYNEKFKDRVTLTA DKSSSTASMHLRSLRSDDTAVYFCKRSLLALDYWGQGTLVTVSS 260

Antibody Sequence Optimization

Variable domain sequences may be analyzed for sequence characteristicsthat may impact antibody function, expression, stability, and/orimmunogenicity. In some cases, such characteristics may include NGresidue pairs. NG residue pairs may be susceptible to asparaginedeamidation, with possible conversion to glutamate and pyroglutamate ina 3:1 ratio over time. These residue pairs may be mutated, for example,to SG or QG pairs to prevent deamidation at these sites. Alternatively,these antibodies may be formulated to reduce deamidation.

In some embodiments, aspartate isomerization sites may be identified andaltered. Aspartate isomerization sites include DG amino acid residuepairs. Aspartic acid at these sites can convert to glutamate andpyroglutamate in a 3:1 ratio over time. DG residue pairs may be mutatedto SG or QG residue pairs to prevent isomerization at these sites.Alternatively, these antibodies may be formulated to reduce deamidation.

In some embodiments, N-terminal glutamine residues may be converted toN-terminal glutamate residues. This may prevent N-terminal pyrolization.

In some embodiments, one or more aggregation-prone patch of amino acidresidues may be mutated. These may include patches having amino acidswith bulky side chains, for example, histidine, phenylalanine, andtryptophan.

In some embodiments, one or more cysteine residues may be mutated toprevent the presence of unpaired cysteines. Unpaired cysteines may bereactive, for example, when accessible to solvent as part of anantibody. In some cases, unpaired cysteine residues may be mutated toserine.

In some embodiments, one or more glycosylation sites (e.g., N-linkedNXS/T sites), acid cleavage sites, and amino acid oxidation sites aremutated to improve antibody production, stability, binding, and/oractivity.

IgG Synthesis

IgG antibodies (e.g. IgG1, IgG2, IgG3 or IgG4) including one or morevariable domain and/or CDR amino acid sequences presented herein (orfragment or variants thereof) may be synthesized for further testingand/or product development. Such antibodies may be produced by insertionof one or more segments of cDNA encoding desired amino acid sequencesinto expression vectors suited for IgG production. Expression vectorsmay include mammalian expression vectors suitable for IgG expression inmammalian cells. Mammalian expression of IgGs may be carried out toensure that antibodies produced include modifications (e.g.glycosylation) characteristic of mammalian proteins and/or to ensurethat antibody preparations lack endotoxin and/or other contaminants thatmay be present in protein preparations from bacterial expressionsystems.

Immunogenic Hosts

In some embodiments, glycan-interacting antibodies of the presentinvention may be developed through the use of non-human animals as hostsfor immunization, referred to herein as “immunogenic hosts”. In someembodiments, immunogenic hosts are mammals. In some embodiments,immunogenic hosts are transgenic knockout mice. Antigens having targetsites and/or epitope targets of glycan-interacting antibodies may beused to contact immunogenic hosts in order to stimulate an immuneresponse and produce antibodies in the immunogenic host thatspecifically bind the target sites and/or epitope targets present on theantigens introduced.

According to some methods of the present invention, the development ofanti-STn antibodies may include immunizing mice that have had the Cmahgene disrupted. Such mutations may result in more human-like physiologyin that Neu5Gc, the immunogenic, non-human form of sialic acid, is nolonger produced in such mice. Also provided is a Cmah^(-/-) myeloma cellfor producing a hybridoma that is free of Neu5Gc expression, forproduction of a GcSTn monoclonal antibody either by reducing the amountof recoverable anti-GcSTn or the hybridoma will begin to die due toantibody binding back to the hybridoma. Other genes can be knocked outin the background of Cmah^(-/-) myeloma cells. For example, thealpha1,3-galactosyltransferase gene, which encodes an enzyme criticalfor the formation of an epitope highly-immunogenic to humans (Chung,C.H. et al., Cetuximab-induced anaphylaxis and IgE specific forgalactose-alpha-1,3-galactose. N Engl J Med. 2008 Mar13;358(11):1109-17), can be knocked out in the background of Cmah^(-/-)myeloma cells.

According to other methods of the present invention, wild type mice maybe used for immunization. Such methods may sometimes be favorable forthe production of antibodies that interact with AcSTn or pan-STnepitopes. In some cases, immune responses in wild type mice may be morerobust.

Antibodies produced through immunization may be isolated from serum ofthe immunogenic hosts. Antibody producing cells from the immunogenichosts may also be used to generate cell lines that produce the desiredantibody. In some embodiments, screening for antibodies and/or antibodyproducing cells from the immunogenic host may be carried out through theuse of enzyme-linked immunosorbent assays (ELISAs) and/or glycan arrays.

Adjuvants

Immunization of immunogenic hosts with antigens described herein mayinclude the use of one or more adjuvants. Adjuvants may be used toelicit a higher immune response in such immunogenic hosts. As such,adjuvants used according to the present invention may be selected basedon their ability to affect antibody titers.

In some embodiments, water-in-oil emulsions may be useful as adjuvants.Water-in-oil emulsions may act by forming mobile antigen depots,facilitating slow antigen release and enhancing antigen presentation toimmune components. Freund’s adjuvant may be used as complete Freund’sadjuvant (CFA), which includes mycobacterial particles that have beendried and inactivated, or as incomplete Freund’s adjuvant (IFA), lackingsuch particles. Other water-in-oil-based adjuvants may includeEMULSIGEN® (MVP Technologies, Omaha, NE). EMULSIGEN® includes micronsized oil droplets that are free from animal-based components. It may beused alone or in combination with other adjuvants, including, but notlimited to aluminum hydroxide and CARBIGEN™ (MVP Technologies, Omaha,NE).

In some embodiments, TITERMAX® adjuvant may be used. TITERMAX® isanother water-in-oil emulsion that includes squalene as well as sorbitanmonooleate 80 (as an emulsifier) and other components. In some cases,TITERMAX® may provide higher immune responses, but with decreasedtoxicity toward immunogenic hosts.

Immunostimmulatory oligonucleotides may also be used as adjuvants. Suchadjuvants may include CpG oligodeoxynucleotide (ODN). CpG ODNs arerecongnized by Toll-like receptor 9 (TLR9) leading to strongimmunostimulatory effects. Type C CpG ODNs induce strong IFN-αproduction from plasmacytoid dendritic cell (pDC) and B cell stimulationas well as IFN-γ production from T-helper (T_(H)) cells. CpG ODNadjuvant has been shown to significantly enhance pneumococcalpolysaccharide (19F and type 6B)-specific IgG2a and IgG3 in mice. CpGODN also enhanced antibody responses to the protein carrier CRM197,particularly CRM197-specific IgG2a and IgG3 (Chu et al., InfectionImmunity 2000, vol 68(3):1450-6). Additionally, immunization of agedmice with pneumococcal capsular polysaccharide serotype 14 (PPS14)combined with a CpG-ODN restored IgG anti-PPS14 responses to young adultlevels (Sen et al., Infection Immunity, 2006, 74(3):2177-86). CpG ODNsused according to the present invention may include class A, B or CODNs. In some embodiments, ODNs may include any of those availablecommercially, such as ODN-1585, ODN-1668, ODN-1826, ODN-2006, ODN-2007,ODN-2216, ODN-2336, ODN-2395 and/or ODN-M362, each of wich may bepurchased, for example, from InvivoGen, (San Diego, CA). In some cases,ODN-2395 may be used. ODN-2395 is a class C CpG ODN that specificallystimulated human as well as mouse TLR9. These ODNs includephosphorothioate backbones and CpG palindromic motifs.

In some embodiments, immune stimulating complexes (ISCOMs) may be usedas adjuvants. ISCOMs are spherical open cage-like structures (typically40 nm in diameter) that are spontaneously formed when mixing togethercholesterol, phospholipids and Quillaia saponins under a specificstoichiometry. ISCOM technology is proven for a huge variety of antigensfrom large glycoproteins such as gp340 from Epstein-Barr virus (a 340kDa antigen consisting of 80% carbohydrates) down to carrier-conjugatedsynthetic peptides and small haptens such as biotin. Some ISCOMs arecapable of generating a balanced immune response with both T_(H1) andT_(H2) characteristics. Immune response to ISCOMs is initiated indraining lymph nodes, but is efficiently relocated to the spleen, whichmakes it particularly suitable for generating monoclonal antibodies aswell. In some embodiments, the ISCOM adjuvant AbISCO-100 (Isconova,Uppsala, Sweden) may used. AbISCO-100 is a saponin-based adjuvantspecifically developed for use in immunogenic hosts, such as mice, thatmay be sensitive to other saponins.

According to embodiments of the present invention, adjuvant componentsof immunization solutions may be varied in order to achieve desiredresults. Such results may include modulating the overall level of immuneresponse and/or level of toxicitiy in immunogenic hosts.

Antibody Sequence and Structural Analysis and Optimization

In some embodiments, antibodies of the present invention may besubjected to sequence analysis and/or structural analysis wherein theyare analyzed for characteristics that may affect antibody chemistry,affinity, specificity, protein folding, stability, manufacturing,expression, and/or immunogenicity (i.e., immune reactions in subjectsbeing treated with such antibodies). Such analysis may includecomparisons between antibodies binding to the same or similar epitopes.

Antibodies sequences of antibodies binding to the same epitope may beanalyzed for variation in light and/or heavy chain sequences. Suchanalysis may include germline sequences and/or CDR sequences.Information obtained from such analysis may be used to identify (andoptionally to modify, delete, replace or repair) conserved amino acidresidues; conserved segments of amino acids; amino acid positions withconserved side chain characteristics; conserved CDR lengths; and otherfeatures conserved among antibodies binding to the same epitope. Thisinformation may be used to design variants or to inform antibodyoptimization procedures to improve antibody affinity, specificity,protein folding, stability, manufacturing, expression and/orimmunogenicity.

Sequence analysis may include aligning two or more antibodies that bindto the same or similar epitopes to identify similarities. Such analysismay compare the sequence and/or length of antibody regions (e.g., CDRs,variable domains, germline segments). Amino acid insertions, amino aciddeletions, and substitutions may be identified and assessed. Sequencedifferences may be compared against antibody affinity and/orspecificity.

In some cases, sequence analyses are conducted to identify (andoptionally to modify, delete, replace or repair) one or more of unpairedcysteines or irregular disulfides; glycosylation sites (e.g., N-linkedNXS/T sites); acid cleavage sites, amino acid oxidation sites,conformity with mouse germline sequences; asparagine deamidation sites;aspartate isomerization sites; N-terminal pyroglutamate formation sites;and aggregation-prone patches in CDRs.

In some cases, the present invention provides sequence analysis-informedvariants of antibodies presented herein. As used herein, the term“sequence analysis-informed variant” refers to an antibody variant thathas been modified based on one or more conclusions derived from antibodysequence analysis. In some cases, antibodies of the invention may bemodified to produce antibody variants that include modifications to oneor more of antibody affinity, specificity, protein folding, stability,manufacturing, expression and/or immunogenicity.

Some sequence analysis-informed variants include one or more CDR lengthmodification. CDR length modified antibodies may include one or moreadded or deleted amino acids in one or more CDRs relative to an originalantibody sequence. In some cases, sequence analysis-informed variantsmay include a substitution of one or more CDRs with one or more CDRsderived from another antibody (e.g., an antibody binding to the same orsimilar epitope). In some cases, sequence analysis-informed variants mayinclude a substitution of a heavy or light chain variable domain fromanother antibody (e.g., an antibody binding to the same or similarepitope). Sequence analysis-informed variants may include modificationsto one or more germline genes that the antibody is expressed from. Suchmodifications may include point mutations, regional mutations,insertional mutations or deletional mutations. In some case, germlinegene modifications are carried out to move CDRs from one known germlinegene to another. Sequence analysis-informed variants may include othervariants described herein, including, but not limited to scFvs,monobodies, diabodies, intrabodies, CARs, antibody mimetics, etc.

In some embodiments, sequence and/or structural analysis may be used toinform the construction of antibody fragment display libraries(including, but not limited to scFv libraries, phage display libraries,and yeast display libraries). In one example, sequence alignment may becarried out to align two or more antibodies with a common antigen orepitope and amino acid residues may be identified that are conservedamong the aligned antibodies or that are variable among the alignedantibodies. In such cases, antibody fragment display libraries may beconstructed such that variability among library members is primarilylimited to the variable amino acids identified in the sequence analysis.In some cases, such libraries may be used to identify variants withaltered affinity and/or specificity for a target antigen (e.g., STn) ora specific epitope of the target antigen (e.g., the epitopes recognizedby Group 1, 2, 3 and 4 antibodies as described in Example 1,hereinbelow).

In some embodiments, antibodies of the invention may be modified toremove, replace or otherwise eliminate one or more unpaired cysteineresidues. In some cases, unpaired cysteine residues may be reactive andin some cases may affect antibody affinity and/or specificity.Accordingly, some antibodies of the invention have been modified toeliminate unpaired cysteine residues. In some cases, such variants mayhave modified epitope specificity and/or affinity. In some cases,modification of unpaired cysteine residues may alter antibody folding.In some cases, these variants include a substitution or deletion of oneor more cysteine residues. In some cases, these variants include one ormore additional amino acid residues (including, but not limited to, theaddition of one or more cysteine residues) to prevent or reduceundesired effects from unpaired cysteine residues. In some cases,cysteine residues are replaced with an amino acid having a hydrophobicside chain (e.g., tyrosine, alanine, valine, isoleucine, leucine,methionine, phenylalanine or tryptophan).

Antibody Testing and Characterization

Antibodies described herein may be tested and/or characterized using avariety of methods. Such methods may be used to determine a variety ofcharacteristics that may include, but are not limited to, antibodyaffinity; specificity; and activity (e.g., activation or inhibition ofcellular signaling pathways or other cellular or biological activities).Antibody testing may further include testing in vivo (e.g., in animaland/or human studies) for one or more of toxicity, therapeutic effect,pharmacodynamics, pharmacokinetics, absorption, deposition, metabolism,and excretion. Testing in animals may include, but is not limited to,testing in mice, rats, rabbits, guinea pigs, pigs, primates (e.g.,cynomolgus monkeys), sheep, goats, horses, and cattle.

Cell-based Assays

In some embodiments, antibodies of the present invention may be testedor characterized through the use of one or more cell-based assays. Suchcell-based assays may be carried out in vitro with cells in culture. Insome cases, cell-based assays may be carried out in vivo. Examples ofcell-based in vivo assays include tumor models in which tumor cells areinjected or otherwise introduced into a host.

In some cases, cells used in cell-based assays may express one or moretarget glycans recognized by one or more antibodies of the invention.Such glycans may be naturally expressed by such cells or, alternatively,cells may be induced to express one or more glycans desired for purposesof a particular assay. Induced expression may be through one or moretreatments that upregulate expression of glycosylated proteins orenzymes that regulate glycosylation. In other cases, induced expressionmay include transfection, transduction, or other form of introduction ofone or more genes or transcripts for the endogenous expression of one ormore glycosylated proteins or enzymes involved in regulation ofglycosylation.

In some cases, cell-based assays used herein may include the use ofcancer cells. Many cancer cell lines are available for experiments totest antibodies of the invention. Such cells may express target glycanor may be induced to express target glycans. Additionally, cancer celllines may be used to test antibodies of the invention, where the cancercell lines are representative of cancer stem cells. Cancer stem cell(CSC) cell lines may be isolated or differentiated from cancer cellsgrown in culture (e.g., through sorting based on markers specific forcancer stem cells). Cell lines used in cell-based assays may include,but are not limted to breast, colon, ovary, lymphocyte, bone marrow, andskin cell lines. Specific cell lines may include, but are not limited toSNU-16 cells, LS-174T cells, MC38 cells, TOV-112D cells, TOV-21G cells,Jurkate E6.1 cells, K-562 cells, B16-F0 cells, B16-F10 cells, LS180cells, COLO205 cells, TB4 cells, HT29 cells, Panc1 cells, HPAC cells,HPAFII cells, RKO cells, SW480 cells, and SNU-C2A cells.

In some embodiments, ovarian cancer cell lines may be used. Such celllines may include, but are not limited to SKOV3, OVCAR3, OV90 and A2870cell lines. In some cases, CSC cells may be isolated from these celllines by isolating cells expressing CD44 and/or CD133 cell markers.

OVCAR3 cells were first established using malignant ascites obtainedfrom a patient suffering from progressive ovarian adenocarcinoma(Hamilton, T.C. et al., 1983. Cancer Res. 43: 5379-89). Cancer stem cellpopulations may be isolated from OVCAR3 cell cultures through selectionbased on specific cell surface markers such as CD44 (involved in celladhesion and migration), CD133 and CD117 (Liang, D. et al., 2012. BMCCancer. 12: 201, the contents of which are herein incorporated byreference in their entirety). OV90 cells are epithelial ovarian cancercells that were similarly derived from human ascites (see U.S. Pat. No.5,710,038). OV-90 cells may also express CD44 when activated (Meunier,L. et al., 2010. Transl Oncol. 3(4): 230-8).

In some embodiments, cell lines derived from gastric cancers may beused. Such cell lines may include, but are not limited to SNU-16 cells(see description in Park J.G. et al., 1990. Cancer Res. 50: 2773-80, thecontents of which are herein incorporated by reference in theirentirety). SNU-16 cells express STn naturally, but at low levels.

In some embodiments, methods of the present disclosure include methodsof characterizing glycan-interacting antibodies by contacting colorectalcells with glycan-interacting antibodies and evaluating antibody bindingto the cells, antibody internalization into the cells, and/or antibodykilling of the cells. According to some such methods, the colorectalcells may be derived from a colorectal cell line grown in vitro (e.g.,propagated through cell culture). In some cases, colorectal cell linesare derived from a tumor. In other embodiments, colorectal cell linesmay be derived from a tumor formed using a xenograft animal model (e.g.,a xenograft mouse model). Colorectal cells used for characterizingglycan-interacting antibodies may be from a patient (e.g., a patienttumor). Methods of characterizing glycan-interacting antibodies mayinclude the use of tissue micro arrays, including those having one ormore colorectal cells.

Characterizing glycan-interacting antibodies with colorectal cells mayinclude evaluating binding between such antibodies and cells bydetermining the EC50 of binding of the glycan-interacting antibody tothe colorectal cell. The EC₅₀ may be determined by using one or more offlow cytometry analysis and ELISA analysis. In some embodiments,characterizing glycan-interacting antibodies with colorectal cells mayinclude evaluating the killing of such cells by glycan-interactingantibodies. This may be carried out by treating colorectal cells withglycan-interacting antibodies and using a cell viability assay todetermine the percentage of cells killed by the treatment. In somecases, evaluating killing of colorectal cells by glycan-interactingantibodies includes determining the IC₅₀ for glycan-interacting antibodykilling of colorectal cells. In some cases, the antibodies may beconjugated with a cytotoxic agent (e.g., MMAE or MMAF).

Glycan Arrays

In some embodiments, glycan-interacting antibodies of the presentinvention may be developed through the use of glycan arrays. As usedherein, the term “glycan array” refers to a tool used to identify agentsthat interact with any of a number of different glycans linked to thearray substrate. In some embodiments, glycan arrays include a number ofchemically-synthesized glycans, referred to herein as “glycan probes”.In some embodiments, glycan arrays include at least 2, at least 5, atleast 10, at least 20, at least 30, at least 40, at least 50, at least60, at least 70, at least 80, at least 90, at least 100, at least 150,at least 350, at least 1000 or at least 1500 glycan probes. In someembodiments, glycan arrays may be customized to present a desired set ofglycan probes. In some embodiments, glycan probes may be attached to thearray substrate by a linker molecule. Such linkers may include moleculesincluding, but not limited to —O(CH₂)₂CH₂)NH₂ andO(CH₂)₃NHCOCH₂(OCH₂CH₂)₆NH₂.

In some embodiments, a glycan array has more than 70chemically-synthesized glycans, most of which are presented as Neu5Acand Neu5Gc-containing glycan pairs. Some examples of glycan probes mayinclude: Neu5Ac-α-2-6-GalNAc (AcSTn); Neu5Gc-α-2-6-GalNAc (GcSTn);Neu5,9Ac2-α-2,6-GalNAc; Neu9Ac5Gc-α-2,6-GalNAc, and GalNAc (Tn). Theantibody binding specificity to AcSTn vs. GcSTn can be determined usingthe array or other methods of determining specificity known in the art.In addition, the binding profile of antibodies to O-acetylated STn canbe determined. The loss of O-acetylation on STn is relevant to cancer ascancer-associated expression correlates with increased STn recognitionby antibodies (Ogata, S. et al., Tumor-associated sialylated antigensare constitutively expressed in normal human colonic mucosa. Cancer Res.1995 May 1;55(9):1869-74) In some cases, glycan arrays may be used todetermine recognition of STn vs. Tn.

Antibody Fragment Display Library Screening Techniques

In some embodiments, antibodies of the present invention may be producedand/or optimized using high throughput methods of discovery. Suchmethods may include any of the display techniques (e.g. display libraryscreening techniques) disclosed in International Patent Application No.WO2014074532, the contents of which are herein incorporated by referencein their entirety. In some embodiments, synthetic antibodies may bedesigned, selected or optimized by screening target antigens usingdisplay technologies (e.g. phage display technologies). Phage displaylibraries may include millions to billions of phage particles, eachexpressing unique antibody fragments on their viral coats. Suchlibraries may provide richly diverse resources that may be used toselect potentially hundreds of antibody fragments with diverse levels ofaffinity for one or more antigens of interest (McCafferty, et al., 1990.Nature. 348:552-4; Edwards, B.M. et al., 2003. JMB. 334: 103-18;Schofield, D. et al., 2007. Genome Biol. 8, R254 and Pershad, K. et al.,2010. Protein Engineering Design and Selection. 23:279-88; the contentsof each of which are herein incorporated by reference in theirentirety). Often, the antibody fragments present in such librariesinclude scFv antibody fragments that include a fusion protein of V_(H)and V_(L) antibody domains joined by a flexible linker. In some cases,scFvs may contain the same sequence with the exception of uniquesequences encoding variable loops of the complementarity determiningregions (CDRs). In some cases, scFvs are expressed as fusion proteins,linked to viral coat proteins (e.g. the N-terminus of the viral pIIIcoat protein). V_(L) chains may be expressed separately for assemblywith V_(H) chains in the periplasm prior to complex incorporation intoviral coats. Precipitated library members may be sequenced from thebound phage to obtain cDNA encoding desired scFvs. Such sequences may bedirectly incorporated into antibody sequences for recombinant antibodyproduction, or mutated and utilized for further optimization through invitro affinity maturation.

Development of Cytotoxic Antibodies

In some embodiments, antibodies of the present invention may be capableof inducing antibody-dependent cell-mediated cytotoxicity (ADCC) and/orantibody-dependent cell phagocytosis (ADCP). ADCC is an immune mechanismwhereby cells are lysed as a result of immune cell attack. Such immunecells may include CD56+ cells, CD3- natural killer (NK) cells, monocytesand neutrophills (Strohl, W.R. Therapeutic Antibody Engineering.Woodhead Publishing, Philadelphia PA. 2012. Ch. 8, p186, the contents ofwhich are herein incorporated by reference in their entirety).

In some cases, antibodies of the present invention may be engineered toinclude a given isotype depending on whether or not ADCC or ADCP isdesired upon antibody binding. Such antibodies, for example, may beengineered according to any of the methods disclosed by Alderson, K.L.et al., J Biomed Biotechnol. 2011. 2011:379123). In the case of mouseantibodies, different isotypes of antibodies are more effective atpromoting ADCC. IgG2a, for example, is more effective at inducing ADCCthan is IgG2b. Some antibodies of the present invention, including mouseIgG2b antibodies may be reengineered to be IgG2a antibodies. Suchreengineered antibodies may be more effective at inducing ADCC uponbinding cell-associated antigens. In some embodiments, antibodies arereengineered by modifying or introducing one or more post-translationalmodifications to improve ADCC and/or CDC biological activity.

In some embodiments, genes encoding variable regions of antibodiesdeveloped according to methods of the present invention may be clonedinto mammalian expression vectors encoding human Fc regions. Such Fcregions may be Fc regions from human IgG1κ. IgG1κ Fc regions may includeamino acid mutations known to enhance Fc-receptor binding andantibody-dependent cell-mediated cytotoxicity (ADCC).

In some embodiments, antibodies of the invention may be developed forantibody-drug conjugate (ADC) therapeutic applications. ADCs areantibodies in which one or more cargo (e.g., therapeutic agents) areattached [e.g. directly or via linker (e.g. a cleavable linker or anon-cleavable linker)]. ADCs are useful for delivery of therapeuticagents (e.g., drugs or cytotoxic agents) to one or more target cells ortissues (Panowski, S. et al., 2014. mAbs 6:1, 34-45). In some cases,ADCs may be designed to bind to a surface antigen on a targeted cell.Upon binding, the entire antibody-antigen complex may be internalizedand directed to a cellular lysosome. ADCs may then be degraded,releasing the bound cargo. Where the cargo is a cytotoxic agent, thetarget cell will be killed or otherwise disabled. Cytotoxic agents mayinclude, but are not limited to cytoskeletal inhibitors [e.g. tubulinpolymerization inhibitors such as maytansines or auristatins (e.g.monomethyl auristatin E [MMAE] and monomethyl auristatin F [MMAF])] andDNA damaging agents (e.g. DNA polymerization inhibitors such ascalcheamicins and duocarmycins).

In some embodiments, antibodies of the invention may be tested for theirability to promote cell death when developed as ADCs. Cell viabilityassays may be performed in the presence and absence of secondaryantibody-drug conjugates. Antibodies with potent cell growth inhibitionmay then be used to design direct antibody-drug conjugates (ADCs). Theuse of such secondary antibody-drug conjugates in cell-based cytotoxicassays may allow for quick pre-screening of many ADC candidates. Basedon such assays, an unconjugated antibody candidate is directly added tocells in the presence of a secondary antibody that is conjugated to oneor more cytotoxic agents (referred to herein as a 2°ADC).Internalization of the antibody/2°ADC complex into cells that express ahigh density of the targeted antigen can achieve a dose-dependent drugrelease within the cells, causing a cytotoxic effect to kill the cells(e.g., tumor cells), while cells expressing a low density of thetargeted antigen are not affected (e.g., normal cells).

ADCs of the invention may be designed to target cancer cells. Such ADCsmay include antibodies directed to one or more tumor-associatedcarbohydrate antigen (TACA). In some cases, ADCs of the invention areanti-STn antibodies.

Development of Chimeric Antigen Receptors

In some embodiments, antibody sequences of the invention may be used todevelop a chimeric antigen receptor (CAR). CARs are transmembranereceptors expressed on immune cells that facilitate recognition andkilling of target cells (e.g. tumor cells). CARs typically include threebasic parts. These include an ectodomain (also known as the recognitiondomain), a transmembrane domain and an intracellular (signaling) domain.Ectodomains facilitate binding to cellular antigens on target cells,while intracellular domains typically include cell signaling functionsto promote the killing of bound target cells. Further, they may have anextracellular domain with one or more antibody variable domainsdescribed herein or fragments thereof. CARs of the invention alsoinclude a transmembrane domain and cytoplasmic tail. CARs may bedesigned to include one or more segments of an antibody, antibodyvariable domain and/or antibody CDR, such that when such CARs areexpressed on immune effector cells, the immune effector cells bind andclear any cells that are recognized by the antibody portions of theCARs.

Characteristics of CARs include their ability to redirect T-cellspecificity and reactivity toward a selected target in a non-MHC-restricted manner, exploiting the antigen-binding properties ofmonoclonal antibodies. The non-MHC-restricted antigen recognition givesT cells expressing CARs the ability to recognize antigen independent ofantigen processing, thus bypassing a major mechanism of tumor escape.Moreover, when expressed in T-cells, CARs advantageously do not dimerizewith endogenous T cell receptor (TCR) alpha and beta chains.

CARs engineered to target tumors may have specificity for one or moretumor associated carbohydrate antigens (TACAs). In some embodiments,ectodomains of these CARs may include one or more antibody variabledomains or a fragment thereof. In some embodiments, CARs are expressedin T cells, and may be referred to as “CAR-engineered T cells” or“CAR-Ts”. CAR-Ts may be engineered with CAR ectodomains having one ormore antibody variable domains.

Structural Features of Chimeric Antigen Receptors

With gene-transfer technology, T cells can be engineered to stablyexpress antibodies on their surface, conferring a desired antigenspecificity. Chimeric antigen receptors (CARs) combine anantigen-recognition domain of a specific antibody with an intracellulardomain of the CD3-zeta chain or FcγRI protein having T cell activatingproperties into a single chimeric fusion protein. CAR technologyprovides MHC-unrestricted recognition of target cells by T cells.Removal of the MHC restriction of T cells facilitates the use of thesemolecules in any patient, and also, in both CD8⁺ and CD4⁺ T cells,usually restricted to MHC class I or II epitopes, respectively. The useof Ab-binding regions allows T cells to respond to epitopes formed notonly by protein, but also carbohydrate and lipid. This chimeric receptorapproach is especially suited to immunotherapy of cancer, being able tobypass many of the mechanisms by which tumors avoid immunorecognition,such as MHC down-regulation, lack of expression of costimulatorymolecules, CTL resistance, and induction of T cell suppression, andwhere the use of both CD8⁺ CTL and CD4⁺ T cells are best combined foroptimum antitumor efficacy. This approach has been demonstrated to beapplicable to a wide range of tumor antigens, in addition to virusessuch as HIV (Finney, et al., J. Immunology, 2004, 172:104-113).

Although chimeric antigen receptors can trigger T-cell activation in amanner similar to that of endogenous T-cell receptors, in practice, theclinical application of CAR technology has been impeded by inadequate invivo expansion of chimeric antigen receptor T cells. For example, firstgeneration CARs included as their signaling domain the cytoplasmicregion of the CD3ζ or Fc receptor γ chain. These first generation CARswere tested in phase I clinical studies in patients with ovarian cancer,renal cancer, lymphoma, and neuroblastoma, and were found to inducemodest responses, effectively redirecting T cell cytotoxicity butfailing to enable T cell proliferation and survival upon repeatedantigen exposure. The prototypes for second generation CARs involvedreceptors encompassing both CD28 and CD3ζ, and second generation CARshave been tested for treatment of B cell malignancies and other cancers(Sadelain, et al., (2009) Current Opinion in Immunology, 21(2):215-223).Thus, CARs have rapidly expanded into a diverse array of receptors withdifferent functional properties.

More recently, it was discovered that CAR-mediated T-cell responses canbe enhanced with the addition of a costimulatory domain. In preclinicalmodels, the inclusion of the CD137 (4-1BB) signaling domain was found tosignificantly increase antitumor activity and in vivo persistence ofchimeric antigen receptors as compared with inclusion of the CD3-zetachain alone (Porter, et al., N. Engl. J. Med. 2011, 365:725-733).

Thus, in some embodiments of the present disclosure, antibody sequencesof the invention may be used to develop a chimeric antigen receptor(CAR). In some embodiments, CARs are transmembrane receptors expressedon immune cells that facilitate recognition and killing of target cells(e.g. tumor cells).

In many cancers, tumor-specific antigens for targeting have not beendefined, but in B-cell neoplasms, CD19 is an attractive target.Expression of CD19 is restricted to normal and malignant B cells andB-cell precursors. A pilot clinical trial of treatment with autologous Tcells expressing an anti-CD19 chimeric antigen receptor (CART19) wasperformed in patients with advanced, p53-deficient chronic lymphoidleukemia (CLL). The generation of a CD19-specific immune response inbone marrow was demonstrated by temporal release of cytokines andablation of leukemia cells that coincided with peak infiltration ofchimeric antigen receptor T cells. (Porter, et al., N. Engl. J. Med.2011, 365:725-733).

Further structural features of CARs may include any of those disclosedin several PCT Publications assigned to City of Hope and having thecommon inventor Michael Jensen. For example, PCT Publication WO00/23573describes genetically engineered, CD20-specific redirected T cellsexpressing a cell surface protein having an extracellular domain thatincludes a receptor specific for CD20, an intracellular signalingdomain, and a transmembrane domain. Use of such cells for cellularimmunotherapy of CD20⁺ malignancies and for abrogating any untoward Bcell function. In one embodiment, the cell surface protein is a singlechain FvFc:ζ receptor where Fv designates the VH and VL chains of asingle chain monoclonal antibody to CD20 linked by peptide, Fcrepresents a hinge-CH2-CH3 region of a human IgG1, and ζ represents theintracellular signaling domain of the zeta chain of human CD3. A methodof making a redirected T cell expressing a chimeric T cell receptor byelectroporation using naked DNA encoding the receptor. Similarly, PCTPublication WO02/077029 describes genetically engineered, CD19-specificredirected immune cells expressing a cell surface protein having anextracellular domain that includes a receptor which is specific forCD19, an intracellular signaling domain, and a transmembrane domain. Useof such cells for cellular immunotherapy of CD19⁺ malignancies and forabrogating any untoward B cell function. In one embodiment, the immunecell is a T cell and the cell surface protein is a single chain svFvFc:ζreceptor where scFc designates the VH and VL chains of a single chainmonoclonal antibody to CD19, Fc represents at least part of a constantregion of an IgG1, and zeta represents the intracellular signalingdomain of the T cell antigen receptor complex zeta chain (zeta chain ofhuman CD3). The extracellular domain scFvFc and the intracellular domainzeta are linked by a transmembrane domain such as the transmembranedomain of CD4. A method of making a redirected T cell expressing achimeric T cell receptor by electroportion using naked DNA encoding thereceptor. These chimeric antigen receptors have the ability, whenexpressed in T cells, to redirect antigen recognition based on themonoclonal antibody’s specificity. The design of scFvFc: receptors withtarget specificities for tumor cell-surface epitopes is a conceptuallyattractive strategy to generate antitumor immune effector cells foradoptive therapy as it does not rely on pre-existing anti-tumorimmunity. These receptors are “universal” in that they bind antigen in aMHC independent fashion, thus, one receptor construct can be used totreat a population of patients with antigen positive tumors. City ofHope PCT Publications WO02/088334, WO 2007/059298 and WO 2010/065818describe “zetakines” made up of an extracellular domain that includes asoluble receptor ligand linked to a support region capable of tetheringthe extracellular domain to a cell surface, a transmembrane region andan intracellular signalling domain. Zetakines, when expressed on thesurface of T lymphocytes, direct T cell activity to those specific cellsexpressing a receptor for which the soluble receptor ligand is specific.

Additional features of CARs may include any of those disclosed in twoPCT Publications assigned to University of Texas and having a commoninventor Lawrence Cooper. PCT Publication No. WO 2009/091826 describescompositions that include a human CD19-specific chimeric T cell receptor(or chimeric antigen receptor, CAR) polypeptide (designated hCD19CAR)that includes an intracellular signaling domain, a transmembrane domainand an extracellular domain, the extracellular domain including a humanCD 19 binding region. In another aspect, the CD 19 binding region is anF(ab′)2, Fab′, Fab, Fv or scFv. The intracellular domain may include anintracellular signaling domain of human CD3ζ and may further includehuman CD28 intracellular segment. In certain aspects the transmembranedomain is a CD28 transmembrane domain. PCT Publication No. WO2013/074916 describes methods and compositions for immunotherapyemploying CAR⁺ T cells genetically modified to eliminate expression of Tcell receptor and/or HLA. In particular embodiments, the T cellreceptor-negative and/or HLA-negative T cells are generated using zincfinger nucleases, for example. The CAR⁺ T cells from allogeneic healthydonors can be administered to any patient without causing graft versushost disease (GVHD), acting as universal reagents for off-the-shelftreatment of medical conditions such as cancer, autoimmunity, andinfection.

PCT Publication WO 2011/041093 assigned to the U.S. Department of Healthand Human Services describes anti-vascular endothelial growth factorreceptor-2 chimeric antigen receptors that include an antigen bindingdomain of a KDR-1121 or DC101 antibody, an extracellular hinge domain, aT cell receptor transmembrane domain, and an intracellular T cellreceptor signaling domain, and their use in the treatment of cancer.

PCT Publications WO 2012/079000 and WO 2013/040557, the contents of eachof which are herein incorporated by reference in their entirety, areassigned to University of Pennsylvania and share the common inventorCarl H. June; these publications describe CARs comprising an antigenbinding domain, a transmembrane domain, a costimulatory signalingregion, and a CD3 zeta signaling domain, and methods for generating RNAChimeric Antigen Receptor (CAR) transfected T cells, respectively.

PCT Publication WO2013/126712, also assigned to University ofPennsylvania and sharing the common inventor Carl H. June, describescompositions and methods for generating a persisting population of Tcells exhibiting prolonged exponential expansion in culture that isligand independent and independent of the addition of exogenouscytokines or feeder cells, which are useful for the treatment of cancer.In some embodiments, the antigen binding domain is an anti-cMet bindingdomain. In some embodiments, the antigen binding domain is ananti-mesothelin binding domain. In some embodiments, the antigen bindingdomain is an anti-CD 19 binding domain. The hinge domain is IgG4, thetransmembrane domain is a CD28 transmembrane domain. In someembodiments, the costimulatory signaling region is a CD28 signalingregion. Also provided is a vector comprising a nucleic acid sequenceencoding a chimeric antigen receptor (CAR), and the CAR comprising anantigen binding domain, a hinge domain, a transmembrane domain, acostimulatory signaling region, and a CD3 zeta signaling domain.

PCT Publication WO 2014/039513 assigned to University of Pennsylvaniadescribes compositions and methods for inhibiting one or morediacylglycerol kinase (DGK) isoform in a cell in order to enhance thecytolytic activity of the cell. The cells may be used in adoptive T celltransfer in which, the cell is modified to express a chimeric antigenreceptor (CAR). Inhibition of DGK in T cells used in adoptive T celltransfer increases cytolytic activity of the T cells and thus may beused in the treatment of a variety of conditions, including cancer,infection, and immune disorders.

PCT Publication WO 2014/055771 assigned to University of Pennsylvaniadescribes compositions and methods for treating ovarian cancer.Specifically, the invention relates to administering a geneticallymodified T cell having alpha-folate receptor (FR-alpha) binding domainand CD27 costimulatory domain to treat ovarian cancer. In oneembodiment, the FR-alpha binding domain is said to be fully human,thereby preventing a host immune response.

In some embodiments, CARs of the invention may be engineered to targettumors. Such CARs may have specificity for one or more TACAs. In somecase, ectodomains of these CARs may comprise one or more antibodyvariable domain presented herein or a fragment thereof. In someembodiments, CARs of the invention are expressed in T cells, referred toherein as “CAR-engineered T cells” or “CAR-Ts”. CAR-Ts may be engineeredwith CAR ectodomains having one or more antibody variable domainpresented herein.

Multispecific Antibodies

In some embodiments, antibodies of the present invention may bind morethan one epitope. As used herein, the terms “multibody” or“multispecific antibody” refer to an antibody wherein two or morevariable regions bind to different epitopes. The epitopes may be on thesame or different targets. In certain embodiments, a multi-specificantibody is a “bispecific antibody,” which recognizes two differentepitopes on the same or different antigens.

Bispecific Antibodies

Bispecific antibodies are capable of binding two different antigens.Such antibodies typically comprise antigen-binding regions from at leasttwo different antibodies. For example, a bispecific monoclonal antibody(BsMAb, BsAb) is an artificial protein composed of fragments of twodifferent monoclonal antibodies, thus allowing the BsAb to bind to twodifferent types of antigen. One common application for this technologyis in cancer immunotherapy, where BsMAbs are engineered tosimultaneously bind to a cytotoxic cell (using a receptor like CD3) anda target like a tumor cell to be destroyed.

Bispecific antibodies may include any of those described in Riethmuller,G., 2012. Cancer Immunity. 12:12-18; Marvin, J.S. et al., 2005. ActaPharmacologica Sinica. 26(6):649-58; and Schaefer, W. et al., 2011.PNAS. 108(27):11187-92, the contents of each of which are hereinincorporated by reference in their entirety.

New generations of BsMAb, called “trifunctional bispecific” antibodies,have been developed. These consist of two heavy and two light chains,one each from two different antibodies, where the two Fab regions (thearms) are directed against two antigens, and the Fc region (the foot)comprises the two heavy chains and forms the third binding site.

Of the two paratopes that form the tops of the variable domains of abispecific antibody, one can be directed against a target antigen andthe other against a T-lymphocyte antigen like CD3. In the case oftrifunctional antibodies, the Fc region may additionally binds to a cellthat expresses Fc receptors, like a mactrophage, a natural killer (NK)cell or a dendritic cell. In sum, the targeted cell is connected to oneor two cells of the immune system, which subsequently destroy it.

Other types of bispecific antibodies have been designed to overcomecertain problems, such as short half-life, immunogenicity andside-effects caused by cytokine liberation. They include chemicallylinked Fabs, consisting only of the Fab regions, and various types ofbivalent and trivalent single-chain variable fragments (scFvs), fusionproteins mimicking the variable domains of two antibodies. The furthestdeveloped of these newer formats are the bi-specific T-cell engagers(BiTEs) and mAb2′s, antibodies engineered to contain an Fcabantigen-binding fragment instead of the Fc constant region.

A bispecific, single-chain antibody Fv fragment (Bs-scFv) wassuccessfully used to kill cancer cells. Some human cancers are caused byfunctional defects in p53 that are restored by gene therapy withwild-type p53. Weisbart, et al., describe the construction andexpression of a bispecific single-chain antibody that penetrates livingcolon cancer cells, binds intracellular p53, and targets and restoresits wild type function (Weisbart, et al., Int. J. Oncol. 2004Oct;25(4):1113-8; and Weisbart, et al., Int. J. Oncol. 2004Dec;25(6):1867-73). In these studies, a bispecific, single-chainantibody Fv fragment (Bs-scFv) was constructed from (i) a single-chainFv fragment of mAb 3E10 that penetrates living cells and localizes inthe nucleus, and (ii) a single-chain Fv fragment of a non-penetratingantibody, mAb PAb421 that binds the C-terminal of p53. PAb421 bindingrestores wild-type functions of some p53 mutants, including those ofSW480 human colon cancer cells. The Bs-scFv penetrated SW480 cells andwas cytotoxic, suggesting an ability to restore activity to mutant p53.COS-7 cells (monkey kidney cells with wild-type p53) served as a controlsince they are unresponsive to PAb421 due to the presence of SV40 largeT antigen that inhibits binding of PAb421 to p53. Bs-scFv penetratedCOS-7 cells but was not cytotoxic, thereby eliminating non-specifictoxicity of Bs-scFv unrelated to binding p53. Fv fragments alone werenot cytotoxic, indicating that killing was due to transduction of p53. Asingle mutation in CDR1 of PAb421 VH eliminated binding of the Bs-scFvto p53 and abrogated cytotoxicity for SW480 cells without alteringcellular penetration, further supporting the requirement of PAb421binding to p53 for cytotoxicity (Weisbart, et al., Int. J. Oncol. 2004Oct;25(4):1113-8; and Weisbart, et al., Int. J. Oncol. 2004Dec;25(6):1867-73).

In some embodiments, antibodies of the present invention may bediabodies. Diabodies are functional bispecific single-chain antibodies(bscAb). These bivalent antigen-binding molecules are composed ofnon-covalent dimers of scFvs, and can be produced in mammalian cellsusing recombinant methods. (See, e.g., Mack et al, Proc. Natl. Acad.Sci., 92: 7021-7025, 1995). Few diabodies have entered clinicaldevelopment. An iodine-123-labeled diabody version of the anti-CEAchimeric antibody cT84.66 has been evaluated for pre-surgicalimmunoscintigraphic detection of colorectal cancer in a study sponsoredby the Beckman Research Institute of the City of Hope(Clinicaltrials.gov NCT00647153) (Nelson, A. L., MAbs.2010. Jan-Feb;2(1):77-83).

Using molecular genetics, two scFvs can be engineered in tandem into asingle polypeptide, separated by a linker domain, called a “tandem scFv”(tascFv). TascFvs have been found to be poorly soluble and requirerefolding when produced in bacteria, or they may be manufactured inmammalian cell culture systems, which avoids refolding requirements butmay result in poor yields. Construction of a tascFv with genes for twodifferent scFvs yields a “bispecific single-chain variable fragments”(bis-scFvs). Only two tascFvs have been developed clinically bycommercial firms; both are bispecific agents in active early phasedevelopment by Micromet for oncologic indications, and are described as“Bispecific T-cell Engagers (BiTE).” Blinatumomab is ananti-CD19/anti-CD3 bispecific tascFv that potentiates T-cell responsesto B-cell non-Hodgkin lymphoma in Phase 2. MT110 is ananti-EP-CAM/anti-CD3 bispecific tascFv that potentiates T-cell responsesto solid tumors in Phase 1. Bispecific, tetravalent “TandAbs” are alsobeing researched by Affimed (Nelson, A. L., MAbs.2010. Jan-Feb;2(1):77-83).

Also included are maxibodies (bivalent scFV fused to the amino terminusof the Fc (CH2-CH3 domains) of IgG.

Bispecific T-cell-engager (BiTE) antibodies are designed to transientlyengage cytotoxic T-cells for lysis of selected target cells. Thesetypically include two scFvs (one binding to CD3 on Tcells and onebinding to a target antigen on the surface of a cell being targeted fordestruction). In some embodiments, the two scFvs are joined by a linker.In other embodiments, the two scFvs are different regions on anantibody. The clinical activity of BiTE antibodies corroborates findingsthat ex vivo expanded, autologous T-cells derived from tumor tissue, ortransfected with specific T-cell receptors, have shown therapeuticpotential in the treatment of solid tumors. While these personalizedapproaches prove that T-cells alone can have considerable therapeuticactivity, even in late-stage cancer, they are cumbersome to perform on abroad basis. This is different for cytotoxic T-lymphocyte antigen 4(CTLA-4) antibodies, which facilitate generation of tumor-specificT-cell clones, and also for bi- and tri-specific antibodies thatdirectly engage a large proportion of patients’ T-cells for cancer celllysis. The potential of global T-cell engagement for human cancertherapy by T-cell-engaging antibodies is under active investigation(Baeuerle PA, et al., Current Opinion in Molecular Therapeutics. 2009,11(1):22-30 and Baeuerle PA and Reinhardt C, Cancer Res. 2009, 69(12):4941-4, the contents of each of which are herein incorporated byreference in their entirety).

Third generation molecules include “miniaturized” antibodies. Among thebest examples of mAb miniaturization are the small modularimmunopharmaceuticals (SMIPs) from Trubion Pharmaceuticals. Thesemolecules, which can be monovalent or bivalent, are recombinantsingle-chain molecules containing one V_(L), one V_(H) antigen-bindingdomain, and one or two constant “effector” domains, all connected bylinker domains. Presumably, such a molecule might offer the advantagesof increased tissue or tumor penetration claimed by fragments whileretaining the immune effector functions conferred by constant domains.At least three “miniaturized” SMIPs have entered clinical development.TRU-015, an anti-CD20 SMIP developed in collaboration with Wyeth, is themost advanced project, having progressed to Phase 2 for rheumatoidarthritis (RA). Earlier attempts in systemic lupus erythrematosus (SLE)and B cell lymphomas were ultimately discontinued. Trubion and FacetBiotechnology are collaborating in the development of TRU-016, ananti-CD37 SMIP, for the treatment of CLL and other lymphoid neoplasias,a project that has reached Phase 2. Wyeth has licensed the anti-CD20SMIP SBI-087 for the treatment of autoimmune diseases, including RA, SLEand possibly multiple sclerosis, although these projects remain in theearliest stages of clinical testing. (Nelson, A. L., MAbs.2010. Jan-Feb;2(1):77-83).

Genmab is researching application of their “Unibody” technology, inwhich the hinge region has been removed from IgG4 molecules. While IgG4molecules are unstable and can exchange light-heavy chain heterodimerswith one another, deletion of the hinge region prevents heavychain-heavy chain pairing entirely, leaving highly specific monovalentlight/heavy heterodimers, while retaining the Fc region to ensurestability and extended half-life in vivo. This configuration mayminimize the risk of immune activation or oncogenic growth, as IgG4interacts poorly with FcRs and monovalent unibodies fail topromoteintracellular signaling complex formation. These contentions are,however, largely supported by laboratory, rather than clinical,evidence. Biotecnol is also developing a “miniaturized” mAb, CAB051,which is a “compacted” 100 kDa anti-HER2 antibody in preclinicalresearch (Nelson, A. L., MAbs.2010. Jan-Feb; 2(1):77-83).

Recombinant therapeutics composed of single antigen-binding domains havealso been developed, although they currently account for only 4% of theclinical pipeline. These molecules are extremely small, with molecularweights approximately one-tenth of those observed for full-sized mAbs.Arana and Domantis engineer molecules composed of antigen-bindingdomains of human immunoglobulin light or heavy chains, although onlyArana has a candidate in clinical testing, ART-621, an anti-TNFαmolecule in Phase 2 study for the treatment of psoriasis and rheumatoidarthritis. Ablynx produces “nanobodies” derived from the antigen-bindingvariable heavy chain regions (V_(HHS)) of heavy chain antibodies foundin camels and llamas, which lack light chains. Two Ablynx anti-vonWillebrand Factor nanobodies have advanced to clinical development,including ALX-0081, in Phase 2 development as an intravenous therapy toprevent thrombosis in patients undergoing percutaneous coronaryintervention for acute coronary syndrome, and ALX-0681, a Phase 1molecule for subcutaneous administration intended for both patients withacute coronary syndrome and thrombotic thrombocytopenic purpura (Nelson,A. L., MAbs.2010. Jan-Feb; 2(1):77-83).

Development of Multispecific Antibodies

In some embodiments, antibody sequences of the invention may be used todevelop multispecific antibodies (e.g., bispecific, trispecific, or ofgreater multispecificity). Multispecific antibodies can be specific fordifferent epitopes of a target antigen of the present invention, or canbe specific for both a target antigen of the present invention, and aheterologous epitope, such as a heterologous glycan, peptide or solidsupport material. (See, e.g., WO93/17715; WO92/08802; WO91/00360;WO92/05793; Tutt, A. et al., Trispecific F(ab′)3 derivatives that usecooperative signaling via the TCR/CD3 complex and CD2 to activate andredirect resting cytotoxic T cells. J. Immunol. 1991 Jul 1;147(1):60-9;U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819;and Kostelny, S.A. et al., Formation of a bispecific antibody by the useof leucine zippers. J. Immunol. 1992 Mar 1;148(5):1547-53); U.S. Pat.No. 5,932,448.

Disclosed and claimed in PCT Publication WO2014144573 to MemorialSloan-Kettering Cancer Center are multimerization technologies formaking dimeric multispecific binding agents (e.g., fusion proteinscomprising antibody components) with improved properties overmultispecific binding agents without the capability of dimerization.

Disclosed and claimed in PCT Publication WO2014144357 to Merck PatentGMBH are tetravalent bispecific antibodies (TetBiAbs), and methods ofmaking and methods of using TetBiAbs for diagnostics and for thetreatment of cancer or immune disorders. TetBiAbs feature a second pairof Fab fragments with a second antigen specificity attached to theC-terminus of an antibody, thus providing a molecule that is bivalentfor each of the two antigen specificities. The tetravalent antibody isproduced by genetic engineering methods, by linking an antibody heavychain covalently to a Fab light chain, which associates with itscognate, co-expressed Fab heavy chain.

Disclosed and claimed in PCT Publication WO2014028560 to IBCPharmaceuticals, Inc. are T cell redirecting bispecific antibodies(bsAb), with at least one binding site for a T-cell antigen and at leastone binding site for an antigen on a diseased cell or pathogen, fortreatment of disease. Preferably, this bsAb is an anti-CD3 x anti-CD19bispecific antibody, although antibodies against other T-cell antigensand/or disease-associated antigens may be used. The complex is capableof targeting effector T cells to induce T-cell-mediated cytotoxicity ofcells associated with a disease, such as cancer, autoimmune disease orinfectious disease. The cytotoxic immune response is enhanced byco-administration of interfon-based agents that comprise interferon-α,interferon-bgr; interferon-λ1, interferon-λ2 or interferon-λ3.

Disclosed and claimed in PCT Publication WO2013092001 to Synimmune GMBHis a bispecific antibody molecule, as well as a method for producing thesame, its use and a nucleic acid molecule encoding the bispecificantibody molecule. In particular is provided an antibody molecule thatis capable of mediating target cell restricted activation of immunecells.

Disclosed and claimed in PCT Publication WO2012007167 is a multispecificmodular antibody specifically binding to at least a glycoepitope and areceptor of the erbB class on the surface of a tumor cell, therebycrosslinking the glycoepitope and the receptor, which antibody hasapoptotic activity effecting cytolysis independent of NK cells.

Disclosed and claimed in PCT Publications WO2012048332 and WO2013055404are meditopes, meditope-binding antibodies, meditope delivery systems,as well as a monoclonal antibody framework binding interface formeditopes, and methods for their use. Specifically, two antibody bindingpeptides, C-QFDLSTRRLK-C (“cQFD”; sequence identification number 1therein; SEQ ID NO: 261 herein) and C-QYNLSSRALK-C (“cQYN”; sequenceidentification number 2 therein; SEQ ID NO: 262 herein) were shown tohave novel mAb binding properties. Also called “meditopes,” cQFD andcQYN were shown to bind to a region of the Fab framework of theanti-EGFR mAb cetuximab and not to bind the complementarity determiningregions (CDRs) that bind antigen. The binding region on the Fabframework is distinct from other framework-binding antigens, such as thesuperantigens Staphylococcal protein A (SpA) (Graille et al., 2000) andPeptostreptococcus magnus protein L (PpL) (Graille et al., 2001).Accordingly, one embodiment disclosed is a framework binding interfacecomprising a framework region of a unique murine-human antibody orfunctional fragment thereof that binds a cyclic meditope.

Exemplary patents and patent publications of interest are: U.S. Pat.Nos. 5,585,089; 5,693,761; and 5,693,762, all filed Jun. 7, 1995 andU.S. Pat. No. 6,180,370, all assigned to Protein Design Labs, Inc.,describe methods for producing, and compositions of, humanizedimmunoglobulins having one or more complementarity determining regions(CDR’s) and possible additional amino acids from a donor immunoglobulinand a framework region from an accepting human immunoglobulin. Eachhumanized immunoglobulin chain is said to usually comprise, in additionto the CDR’s, amino acids from the donor immunoglobulin framework thatare, e.g., capable of interacting with the CDRs to effect bindingaffinity, such as one or more amino acids which are immediately adjacentto a CDR in the donor immunoglobulin or those within about about 3 Å aspredicted by molecular modeling. The heavy and light chains may each bedesigned by using any one or all of various position criteria. Whencombined into an intact antibody, the humanized immunoglobulins of thepresent invention is said to be substantially non-immunogenic in humansand retain substantially the same affinity as the donor immunoglobulinto the antigen, such as a protein or other compound containing anepitope.

U.S. Pat. No. 5,951,983, assigned to Universite Catholique De Louvainand Bio Transplant, Inc., describes a humanized antibody againstT-lymphocytes. Framework regions from a human V kappa gene designated asHUM5400 (EMBL accession X55400) and from the human antibody clone Amu5-3 (GenBank accession number U00562) are set forth therein.

U.S. Pat. No. 5,091,513, to Creative Biomolecules, Inc., describes afamily of synthetic proteins having affinity for a preselected antigen.The proteins are characterized by one or more sequences of amino acidsconstituting a region which behaves as a biosynthetic antibody bindingsite (BABS). The sites comprise 1) non-covalently associated ordisulfide bonded synthetic V_(H) and V_(L) dimers, 2) V_(H)-V_(L) orV_(L)-V_(H) single chains wherein the V_(H) and V_(L) are attached by apolypeptide linker, or 3) individuals V_(H) or V_(L) domains. Thebinding domains comprise linked CDR and FR regions, which may be derivedfrom separate immunoglobulins. The proteins may also include otherpolypeptide sequences which function, e.g., as an enzyme, toxin, bindingsite, or site of attachment to an immobilization media or radioactiveatom. Methods are disclosed for producing the proteins, for designingBABS having any specificity that can be elicited by in vivo generationof antibody, and for producing analogs thereof.

U.S. Pat. No. 8,399,625, to ESBATech, an Alcon Biomedical Research Unit,LLC, describes antibody acceptor frameworks and methods for graftingnon-human antibodies, e.g., rabbit antibodies, using a particularly wellsuited antibody acceptor framework.

Intrabodies

In some embodiments, antibodies of the present invention may beintrabodies. Intrabodies are a form of antibody that is not secretedfrom a cell in which it is produced, but instead targets one or moreintracellular proteins. Intrabodies are expressed and functionintracellularly, and may be used to affect a multitude of cellularprocesses including, but not limited to intracellular trafficking,transcription, translation, metabolic processes, proliferative signalingand cell division. In some embodiments, methods described herein includeintrabody-based therapies. In some such embodiments, variable domainsequences and/or CDR sequences disclosed herein are incorporated intoone or more constructs for intrabody-based therapy. For example,intrabodies may target one or more glycated intracellular proteins ormay modulate the interaction between one or more glycated intracellularproteins and an alternative protein.

More than two decades ago, intracellular antibodies againstintracellular targets were first described (Biocca, Neuberger andCattaneo EMBO J. 9: 101-108, 1990). The intracellular expression ofintrabodies in different compartments of mammalian cells allows blockingor modulation of the function of endogenous molecules (Biocca, et al.,EMBO J. 9: 101-108, 1990; Colby et al., Proc. Natl. Acad. Sci. U.S.A.101 : 17616-21, 2004). Intrabodies can alter protein folding,protein-protein, protein-DNA, protein-RNA interactions and proteinmodification. They can induce a phenotypic knockout and work asneutralizing agents by direct binding to the target antigen, bydiverting its intracellular traffic or by inhibiting its associationwith binding partners. They have been largely employed as research toolsand are emerging as therapeutic molecules for the treatment of humandiseases as viral pathologies, cancer and misfolding diseases. The fastgrowing bio-market of recombinant antibodies provides intrabodies withenhanced binding specificity, stability and solubility, together withlower immunogenicity, for their use in therapy (Biocca, abstract inAntibody Expression and Production Cell Engineering Volume 7, 2011, pp.179-195).

In some embodiments, intrabodies have advantages over interfering RNA(iRNA); for example, iRNA has been shown to exert multiple non-specificeffects, whereas intrabodies have been shown to have high specificityand affinity of to target antigens. Furthermore, as proteins,intrabodies possess a much longer active half-life than iRNA. Thus, whenthe active half-life of the intracellular target molecule is long, genesilencing through iRNA may be slow to yield an effect, whereas theeffects of intrabody expression can be almost instantaneous. Lastly, itis possible to design intrabodies to block certain binding interactionsof a particular target molecule, while sparing others.

Development of Intrabodies

Intrabodies are often single chain variable fragments (scFvs) expressedfrom a recombinant nucleic acid molecule and engineered to be retainedintracellularly (e.g., retained in the cytoplasm, endoplasmic reticulum,or periplasm). Intrabodies may be used, for example, to ablate thefunction of a protein to which the intrabody binds. The expression ofintrabodies may also be regulated through the use of inducible promotersin the nucleic acid expression vector comprising the intrabody.Intrabodies may be produced using methods known in the art, such asthose disclosed and reviewed in: (Marasco et al., 1993 Proc. Natl. Acad.Sci. USA, 90: 7889-7893; Chen et al., 1994, Hum. Gene Ther. 5:595-601;Chen et al., 1994, Proc. Natl. Acad. Sci. USA, 91: 5932-5936;Maciejewski et al., 1995, Nature Med., 1: 667-673; Marasco, 1995,Immunotech, 1: 1-19; Mhashilkar, et al., 1995, EMBO J. 14: 1542-51; Chenet al., 1996, Hum. Gene Therap., 7: 1515-1525; Marasco, Gene Ther.4:11-15, 1997; Rondon and Marasco, 1997, Annu. Rev. Microbiol.51:257-283; Cohen, et al., 1998, Oncogene 17:2445-56; Proba et al.,1998, J. Mol. Biol. 275:245-253; Cohen et al., 1998, Oncogene17:2445-2456; Hassanzadeh, et al., 1998, FEBS Lett. 437:81-6; Richardsonet al., 1998, Gene Ther. 5:635-44; Ohage and Steipe, 1999, J. Mol. Biol.291:1119-1128; Ohage et al., 1999, J. Mol. Biol. 291:1129-1134; Wirtzand Steipe, 1999, Protein Sci. 8:2245-2250; Zhu et al., 1999, J.Immunol. Methods 231:207-222; Arafat et al., 2000, Cancer Gene Ther.7:1250-6; der Maur et al., 2002, J. Biol. Chem. 277:45075-85; Mhashilkaret al., 2002, Gene Ther. 9:307-19; and Wheeler et al., 2003, FASEB J.17: 1733-5; and references cited therein). In particular, a CCR5intrabody has been produced by Steinberger et al., 2000, Proc. Natl.Acad. Sci. USA 97:805-810). See generally Marasco, WA, 1998,“Intrabodies: Basic Research and Clinical Gene Therapy Applications”Springer:New York; and for a review of scFvs, see Pluckthun in “ThePharmacology of Monoclonal Antibodies,” 1994, vol. 113, Rosenburg andMoore eds. Springer-Verlag, New York, pp. 269-315.

In some embodiments, antibody sequences are used to develop intrabodies.Intrabodies are often recombinantly expressed as single domain fragmentssuch as isolated VH and VL domains or as a single chain variablefragment (scFv) antibody within the cell. For example, intrabodies areoften expressed as a single polypeptide to form a single chain antibodycomprising the variable domains of the heavy and light chain joined by aflexible linker polypeptide. Intrabodies typically lack disulfide bondsand are capable of modulating the expression or activity of target genesthrough their specific binding activity. Single chain antibodies canalso be expressed as a single chain variable region fragment joined tothe light chain constant region.

As is known in the art, an intrabody can be engineered into recombinantpolynucleotide vectors to encode sub-cellular trafficking signals at itsN or C terminus to allow expression at high concentrations in thesub-cellular compartments where a target protein is located. Forexample, intrabodies targeted to the endoplasmic reticulum (ER) areengineered to incorporate a leader peptide and, optionally, a C-terminalER retention signal, such as the KDEL amino acid motif. Intrabodiesintended to exert activity in the nucleus are engineered to include anuclear localization signal. Lipid moieties are joined to intrabodies inorder to tether the intrabody to the cytosolic side of the plasmamembrane. Intrabodies can also be targeted to exert function in thecytosol. For example, cytosolic intrabodies are used to sequesterfactors within the cytosol, thereby preventing them from beingtransported to their natural cellular destination.

There are certain technical challenges with intrabody expression. Inparticular, protein conformational folding and structural stability ofthe newly-synthesized intrabody within the cell is affected by reducingconditions of the intracellular environment. In human clinical therapy,there are safety concerns surrounding the application of transfectedrecombinant DNA, which is used to achieve intrabody expression withinthe cell. Of particular concern are the various viral-based vectorscommonly-used in genetic manipulation. Thus, one approach to circumventthese problems is to fuse protein transduction domains (PTD) to scFvantibodies, to create a ‘cell-permeable’ antibody or ‘Transbody.’Transbodies are cell-permeable antibodies in which a proteintransduction domain (PTD) is fused with single chain variable fragment(scFv) antibodies (Heng and Cao, 2005, Med Hypotheses. 64:1105-8).

Upon interaction with a target gene, an intrabody modulates targetprotein function and/or achieves phenotypic/functional knockout bymechanisms such as accelerating target protein degradation andsequestering the target protein in a non-physiological sub-cellularcompartment. Other mechanisms of intrabody-mediated gene inactivationcan depend on the epitope to which the intrabody is directed, such asbinding to the catalytic site on a target protein or to epitopes thatare involved in protein-protein, protein-DNA, or protein-RNAinteractions.

In one embodiment, intrabodies are used to capture a target in thenucleus, thereby preventing its activity within the nucleus. Nucleartargeting signals are engineered into such intrabodies in order toachieve the desired targeting. Such intrabodies are designed to bindspecifically to a particular target domain. In another embodiment,cytosolic intrabodies that specifically bind to a target protein areused to prevent the target from gaining access to the nucleus, therebypreventing it from exerting any biological activity within the nucleus(e.g., preventing the target from forming transcription complexes withother factors).

In order to specifically direct the expression of such intrabodies toparticular cells, the transcription of the intrabody is placed under theregulatory control of an appropriate tumor-specific promoter and/orenhancer. In order to target intrabody expression specifically toprostate, for example, the PSA promoter and/or promoter/enhancer can beutilized (See, for example, U.S. Pat. No. 5,919,652 issued 6 Jul. 1999).

Protein transduction domains (PTDs) are short peptide sequences thatenable proteins to translocate across the cell membrane and beinternalized within the cytosol, through atypical secretory andinternalization pathways. There are a number of distinct advantages thata ‘Transbody’ would possess over conventional intrabodies expressedwithin the cell. For a start, ‘correct’ conformational folding anddisulfide bond formation can take place prior to introduction into thetarget cell. More importantly, the use of cell-permeable antibodies or‘Transbodies’ would avoid the overwhelming safety and ethical concernssurrounding the direct application of recombinant DNA technology inhuman clinical therapy, which is required for intrabody expressionwithin the cell. ‘Transbodies’ introduced into the cell would possessonly a limited active half-life, without resulting in any permanentgenetic alteration. This would allay any safety concerns with regards totheir application in human clinical therapy (Heng and Cao 2005, MedHypotheses. 64:1105-8).

Intrabodies are promising therapeutic agents for the treatment ofmisfolding diseases, including Alzheimer’s, Parkinson’s, Huntington’sand prion diseases, because of their virtually infinite ability tospecifically recognize the different conformations of a protein,including pathological isoforms, and because they can be targeted to thepotential sites of aggregation (both intra- and extracellular sites).These molecules can work as neutralizing agents against amyloidogenicproteins by preventing their aggregation, and/or as molecular shuntersof intracellular traffic by rerouting the protein from its potentialaggregation site (Cardinale, and Biocca, Curr. Mol. Med. 2008, 8:2-11).

Exemplary Patent Publications describing intracellular antibodies orintrabodies are set forth hereinbelow, each of which is incorporated byreference in its entirety.

PCT Publication WO03014960 and U.S. Pat. 7,608,453 granted to Cattaneo,et al., describe an intracellular antibody capture technology method ofidentifying at least one consensus sequence for an intracellularantibody (ICS) comprising the steps of: creating a database comprisingsequences of validated intracellular antibodies (VIDA database) andaligning the sequences of validated intracellular antibodies accordingto Kabat; determining the frequency with which a particular amino acidoccurs in each of the positions of the aligned antibodies; selecting afrequency threshold value (LP or consensus threshold) in the range from70% to 100%; identifying the positions of the alignment at which thefrequency of a particular amino acid is greater than or equal to the LPvalue; and identifying the most frequent amino acid, in the position ofsaid alignment.

PCT Publications WO0054057; WO03077945; WO2004046185; WO2004046186;WO2004046187; WO2004046188; WO2004046189; US Patent ApplicationPublications US2005272107; US2005276800; US2005288492; US2010143939;granted US Patents 7,569,390 and 7,897,347 and granted European PatentsEP1560853; and EP1166121 all assigned to the Medical Research Counciland including inventors Cattaneo, et al., describe intracellularintracellular single domain immunoglobulins, and a method fordetermining the ability of a immunoglobulin single domain to bind to atarget in an intracellular environment, as well as methods forgenerating intracellular antibodies.

PCT Publication WO0235237; U.S. Pat. Application Publication 2003235850and granted European Patent EP1328814 naming Catteneo as an inventor andassigned to S.I.S.S.A. Scuola Internazionale Superiore describe a methodfor the in vivo identification of epitopes of an intracellular antigen.

PCT Publication WO2004046192 and European Patent EP1565558 assigned toLay Line Genomics SPA and naming Catteneo as an inventor describe amethod for isolating intracellular antibodies that disrupt andneutralize an interaction between a protein ligand x and a proteinligand y inside a cell. Also disclosed are a method to identify aprotein ligand x able to bind to a known y ligand using intracellularantibodies able to the interaction between x and y; and a method for theisolation of a set of antibody fragments against a significantproportion of the protein-protein interactions of a given cell(interactome) or against the protein interactions that constitute anintracellular pathway or network.

U.S. Pat. Application Publication 2006034834 and PCT PublicationWO9914353 entitled “Intrabody-mediated control of immune reactions” andassigned to Dana Farber Cancer Institute Inc. name inventors Marasco andMhashilkar are directed to methods of altering the regulation of theimmune system, e.g., by selectively targeting individual or classes ofimmunomodulatory receptor molecules (IRMs) on cells comprisingtransducing the cells with an intracellularly expressed antibody, orintrabody, against the IRMs. In a preferred embodiment the intrabodycomprises a single chain antibody against an IRM, e.g, MHC-1 molecules.

PCT Publication WO2013033420 assigned to Dana Farber Cancer InstituteInc. and Whitehead Biomedical Institute, and naming inventors Bradner,Rahl and Young describes methods and compositions useful for inhibitinginteraction between a bromodomain protein and an immunoglobulin (Ig)regulatory element and downregulating expression of an oncogenetranslocated with an Ig locus, as well as for treating a cancer (e.g.,hematological malignancy) characterized by increased expression of anoncogene which is translocated with an Ig locus. Intrabodies aregenerally described.

PCT Publication WO02086096 and U.S. Pat. Application Publication2003104402 entitled “Methods of producing or identifying intrabodies ineukaryotic cells,” assigned to University of Rochester Medical Centerand naming inventors Zauderer, Wei and Smith describe a high efficiencymethod of expressing intracellular immunoglobulin molecules andintracellular immunoglobulin libraries in eukaryotic cells using atrimolecular recombination method. Further provided are methods ofselecting and screening for intracellular immunoglobulin molecules andfragments thereof, and kits for producing, screening and selectingintracellular immunoglobulin molecules, as well as the intracellularimmunoglobulin molecules and fragments produced using these methods.

PCT Publication WO2013023251 assigned to Affinity Biosciences PTY LTDand naming inventors Beasley, Niven and Kiefel describes polypeptides,such as antibody molecules and polynucleotides encoding suchpolypeptides, and libraries thereof, wherein the expressed polypeptidesthat demonstrate high stability and solubility. In particular,polypeptides comprising paired VL and VH domains that demonstratesoluble expression and folding in a reducing or intracellularenvironment are described, wherein a human scFv library was screened,resulting in the isolation of soluble scFv genes that have identicalframework regions to the human germline sequence as well as remarkablethermostability and tolerance of CDR3 grafting onto the scFv scaffold.

European Patent Application EP2314622 and PCT Publications WO03008451and WO03097697 assigned to Esbatech AG and University of Zuerich andnaming inventors Ewert, Huber, Honneger and Plueckthun describe themodification of human variable domains and provide compositions usefulas frameworks for the creation of very stable and soluble single-chainFv antibody fragments. These frameworks have been selected forintracellular performance and are thus ideally suited for the creationof scFv antibody fragments or scFv antibody libraries for applicationswhere stability and solubility are limiting factors for the performanceof antibody fragments, such as in the reducing environment of a cell.Such frameworks can also be used to identify highly conserved residuesand consensus sequences which demonstrate enhanced solubility andstability.

PCT Publication WO02067849 and U.S. Pat. Application Publication2004047891 entitled “Systems devices and methods for intrabody targeteddelivery and reloading of therapeutic agents” describe systems, devicesand methods for intrabody targeted delivery of molecules. Moreparticularly, some embodiments relate to a reloadable drug deliverysystem, which enables targeted delivery of therapeutic agents to atissue region of a subject, in a localized and timely manner.

PCT Publication WO2005063817 and U.S. Pat. 7,884,054 assigned to AmgenInc. and naming inventors Zhou, Shen and Martin describe methods foridentifying functional antibodies, including intrabodies. In particular,a homodimeric intrabody is described, wherein each polypeptide chain ofthe homodimer comprises an Fc region, an scFv, and an intracellularlocalization sequence. The intracellular localization sequence may causethe intrabody to be localized to the ER or the Golgi. Optionally, eachpolypeptide chain comprises not more than one scFv.

PCT Publication WO2013138795 by Vogan, et al. and assigned to PermeonBiologics Inc. describes cell penetrating compositions for delivery ofintracellular antibodies and antibody-like moieties and methods fordelivering them (referred to herein as “AAM moieties” or “an AAMmoiety”) into a cell. Without being bound by theory, the presentdisclosure is based, at least in part, on the discovery that an AAMmoiety can be delivered into a cell by complexing the AAM moiety with acell penetrating polypeptide having surface positive charge (referred toherein as a “Surf+ Penetrating Polypeptide”). Examples of someapplications of intraphilin technology are also provided

PCT Publication WO2010004432 assigned to the Pasteur Institute describesimmunoglobulins from camelidae (camels, dromedaries, llamas andalpacas), about 50% of which are antibodies devoid of light chain. Theseheavy-chain antibodies interact with the antigen by the virtue of onlyone single variable domain, referred to as VHH(s), VHH domain(s) or VHHantibody (ies). Despite the absence of light chain, these homodimericantibodies exhibit a broad antigen-binding repertoire by enlarging theirhypervariable regions, and can act as a transbody and/or intrabody invitro as well as in vivo, when the VHH domain is directed against anintracellular target.

PCT Publication WO2014106639 describes a method for identifying acellular target involved in a cell phenotype by identifying an intrabodythat can modify a cell phenotype and identifying a direct or indirectcellular target of the intrabody. In particular, intrabodies 3H2-1,3H2-VH and 5H4 are capable of inhibiting the degranulation reaction inmast cells triggered by an allergic stimulus; furthermore, intrabodies3H2-1 and 5H4 directly or indirectly targeted a protein of the ABCF1family and C120RF4 family, respectively. These ABCF1 and C120RF4inhibitors are said to be useful in therapy, in particular for treatingallergic and/or inflammatory conditions.

PCT Publication WO0140276 assigned to Urogenesis Inc. generallydescribes the possibility of inhibition of STEAP (Six TransmembraneEpithelial Antigen of the Prostate) proteins using intracellularantibodies (intrabodies).

PCT Publication WO02086505 assigned to University of Manchester and U.S.Pat. Application Publication US2004115740 naming inventors Simon andBenton describe a method for the intracelular analysis of a targetmolecule, wherein intrabodies are said to be preferred. In oneembodiment, a vector (designated pScFv-ECFP) capable of expressing ananti-MUC1 intrabody coupled to CFP is described.

PCT Publication WO03095641 and WO0143778 assigned to Gene TherapySystems Inc. describe compositions and methods for intracellular proteindelivery, and intrabodies are generally described.

PCT Publication WO03086276 assigned to Selective Genetics Inc. describesa platform technology for the treatment of intracellular infections.Compositions and methods described therein include non-target specificvectors that target infectable cells via linked ligands that bind andinternalize through cell surface receptors/moieties associated withinfection. The vectors comprise exogenous nucleic acid sequences thatare expressed upon internalization into a target cell. Vector associatedligands and nucleic acid molecules may be altered to target differentinfectious agents. In addition, the invention provides methods ofidentifying epitopes and ligands capable of directing internalization ofa vector and capable of blocking viral entry.

PCT Publication WO03062415 assigned to Erasmus University describes atransgenic organism comprising a polynucleotide construct encoding anintracellular antibody which disrupts the catalysis of the production ofthe xenoantigen galactose alpha 1,3 galactose and/or a polynucleotideconstruct which encodes an intracellular antibody which bindsspecifically to a retrovirus protein, such as a PERV particle protein.Cells, tissues and organs of the transgenic organism may be used inxenotransplantation.

PCT Publication WO2004099775 entitled “Means for detecting proteinconformation and applications thereof” describes the use of scFvfragments as conformation-specific antibodies for specifically detectinga conformational protein state, said to have applications as sensors forfollowing in livings cells, upon intracellular expression, the behaviorof endogeneous proteins.

PCT Publication WO2008070363 assigned to Imclone Systems Inc. describesa single domain intrabody that binds to an intracellular protein or toan intracellular domain of an intracellular protein, such as Etk, theendothelial and epithelial tyrosine kinase, which is a member of the Tecfamily of non-receptor tyrosine kinases. Also provided is a method ofinhibiting an intracellular enzyme, and treating a tumor in a patient byadministering the intrabody or a nucleic acid expressing the intrabody.

PCT Publication WO2009018438 assigned to Cornell Research FoundationInc. describes a method of identifying a protein that binds to a targetmolecule and has intracellular functionality, by providing a constructcomprising a DNA molecule encoding the protein which binds to the targetmolecule, with the DNA molecule being coupled to a stall sequence. Ahost cell is transformed with the construct and then cultured underconditions effective to form, within the host cell, a complex of theprotein whose translation has been stalled, the mRNA encoding theprotein, and ribosomes. The protein in the complex is in a properlyfolded, active form and the complex is recovered from the cell. Thismethod can be carried out with a cell-free extract preparationcontaining ribosomes instead of a host cell. The present invention alsorelates to a construct which includes a DNA molecule encoding a proteinthat binds to a target molecule and an SecM stalling sequence coupled tothe DNA molecule. The DNA molecule and the SecM stalling sequence arecoupled with sufficient distance between them to permit expression oftheir encoded protein, within the cell, in a properly folded, activeform. The use of intrabodies is generally described.

PCT Publication WO2014030780 assigned to Mogam Biotech ResearchInstitute describes a method named Tat-associated protein engineering(TAPE), for screening a target protein having higher solubility andexcellent thermostability, in particular, an immunoglobulin variabledomain (VH or VL) derived from human germ cells, by preparing a geneconstruct where the target protein and an antibiotic -resistant proteinare linked to a Tat signal sequence, and then expressing this within E.coli. Also disclosed are human or engineered VH and VL domain antibodiesand human or engineered VH and VL domain antibody scaffolds havingsolubility and excellent thermostability, which are screened by the TAPEmethod. Also provided is a library including random CDR sequences in thehuman or engineered VH or VL domain antibody scaffold screened by theTAPE method, a preparing method thereof, a VH or VL domain antibodyhaving binding ability to the target protein screened by using thelibrary, and a pharmaceutical composition including the domain antibody.

European Patent Application EP2422811 describes an antibody that bindsto an intracellular epitope; such intrabodies comprise at least aportion of an antibody that is capable of specifically binding anantigen and preferably does not contain operable sequences coding forits secretion and thus remains within the cell. In one embodiment, theintrabody comprises a scFv. The scFv polypeptide further comprises apolypeptide linker between the VH and VL domains which enables the scFvto form the desired structure for antigen binding. Also described is aspecific embodiment in which the intrabody binds to the cytoplasmicdomain of an Eph receptor and prevents its signaling (e.g.,autophosphorylation). In another specific embodiment, an intrabody bindsto the cytoplasmic domain of a B-type Ephrin (e.g., EphrinB1, EphrinB2or EphrinB3).

PCT Publication WO2011003896 and European Patent Application EP2275442describe intracellular functional PCNA-Chromobodies made using nucleicacid molecule encoding a polypeptide specifically binding toproliferating cell nuclear antigen (PCNA). Examples of such polypeptidescomprising conservative substitutions of one or more amino acids in oneor two framework regions include

MANVQLNESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSDISPSGAVKAYSDSVKGRFTISRDNAKNRLYLQMNSLTPEDTGEYFCTKVQSPRTRIPAPSSQGTQVTVSS (SEQ ID NO: 263)

and

MANVQLNESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSEISPSGAVKAYSDSVKGRFTISRDNAKNRLYLQMNSLTPEDTGEYFCTKVQSPRTRIPAPSSQGTQVTVSS (SEQ ID NO: 264)

, including the framework regions of the polypeptides. In the examples,the framework regions as well as the CDR regions involved in the bindingof PCNA have been determined.

European Patent Application EP2703485 describes a method for selectingplasma cells or plasmablasts, as well as for producing target antigenspecific antibodies, and novel monoclonal antibodies. In one embodiment,cells expressing intracellular immunoglobulin were identified.

Antibody-Coated Agents

In some embodiments, antibodies or antibody fragments described hereinmay be used to prepare a composition that includes an antibody-coatedagent. As used herein, the term “antibody-coated agent” refers to anyparticle, nanoparticle, molecule, protein, fusion-protein, lipid,liposome, cell membrane, cell, or other structure that includes one ormore surface-associated antibodies or antibody fragments.Antibody-coated agents may target one or more glycans, proteins, cells,tissues, and/or organs based on the specificity of the antibody orantibody fragments used for coating.

Antibody-coated agents may include associated, enclosed, or embeddedcargo. The cargo may be a detectable label. Some cargo may include oneor more therapeutic agent. Such therapeutic agents may include, but arenot limited to drugs, chemotherapeutic agents, and cytotoxic agents.Cytotoxic agents may be used to kill or otherwise disable a cell.Cytotoxic agents may include, but are not limited to cytoskeletalinhibitors [e.g. tubulin polymerization inhibitors such as maytansinesor auristatins (e.g. monomethyl auristatin E [MMAE] and monomethylauristatin F [MMAF])] and DNA damaging agents (e.g. DNA polymerizationinhibitors such as calcheamicins and duocarmycins).

In some embodiments, antibody-coated agents may include nanoparticlescoated with one or more antibodies or antibody fragments describedherein. Such antibody-coated agents may target one or more glycan,including, but not limited to cell-associated glycans. Some suchantibody-coated agents include one or more cytoxic agents.

Proteins and Variants

Glycan-interacting antibodies of the present invention may exist as awhole polypeptide, a plurality of polypeptides or fragments ofpolypeptides, which independently may be encoded by one or more nucleicacids, a plurality of nucleic acids, fragments of nucleic acids orvariants of any of the aforementioned. As used herein, “polypeptide”means a polymer of amino acid residues (natural or unnatural) linkedtogether most often by peptide bonds. The term, as used herein, refersto proteins, polypeptides, and peptides of any size, structure, orfunction. In some instances the polypeptide encoded is smaller thanabout 50 amino acids and the polypeptide is then termed a peptide. Ifthe polypeptide is a peptide, it will be at least about 2, 3, 4, or atleast 5 amino acid residues long. Thus, polypeptides include geneproducts, naturally occurring polypeptides, synthetic polypeptides,homologs, orthologs, paralogs, fragments and other equivalents,variants, and analogs of the foregoing. A polypeptide may be a singlemolecule or may be a multi-molecular complex such as a dimer, trimer ortetramer. They may also include single chain or multichain polypeptidesand may be associated or linked. The term polypeptide may also apply toamino acid polymers in which one or more amino acid residues are anartificial chemical analogue of a corresponding naturally occurringamino acid.

The term “polypeptide variant” refers to molecules which differ in theiramino acid sequence from a native or reference sequence. The amino acidsequence variants may possess substitutions, deletions, and/orinsertions at certain positions within the amino acid sequence, ascompared to a native or reference sequence. Ordinarily, variants willpossess at least about 50% identity (homology) to a native or referencesequence, and preferably, they will be at least about 80%, morepreferably at least about 90% identical (homologous) to a native orreference sequence.

In some embodiments “variant mimics” are provided. As used herein, theterm “variant mimic” is one which contains one or more amino acids whichwould mimic an activated sequence. For example, glutamate may serve as amimic for phosphoro-threonine and/or phosphoro-serine. Alternatively,variant mimics may result in deactivation or in an inactivated productcontaining the mimic, e.g., phenylalanine may act as an inactivatingsubstitution for tyrosine; or alanine may act as an inactivatingsubstitution for serine. The amino acid sequences of theglycan-interacting antibodies of the invention may include naturallyoccurring amino acids and as such may be considered to be proteins,peptides, polypeptides, or fragments thereof. Alternatively, theglycan-interacting antibodies may include both naturally andnon-naturally occurring amino acids.

The term “amino acid sequence variant” refers to molecules with somedifferences in their amino acid sequences as compared to a native orstarting sequence. The amino acid sequence variants may possesssubstitutions, deletions, and/or insertions at certain positions withinthe amino acid sequence. “Native” or “starting” sequence should not beconfused with a wild type sequence. As used herein, a native or startingsequence is a relative term referring to an original molecule againstwhich a comparison may be made. “Native” or “starting” sequences ormolecules may represent the wild-type (that sequence found in nature)but do not have to be the wild-type sequence.

Ordinarily, variants will possess at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, at least 99.5% at least 99.8%, or atleast 99.9% sequence identity as compared to a native sequence.“Sequence identity” as it applies to amino acid sequences or nucleotidesequences is defined as the percentage of residues in the candidatesequence that are identical with the residues in the second sequenceafter aligning the sequences and taking gaps and fragments intoconsideration, if necessary, to achieve the maximum percent sequenceidentity. Calculation of the percent identity of two polymericsequences, for example, can be performed by aligning the two sequencesfor optimal comparison purposes (e.g., gaps can be introduced in one orboth of a first and a second polymeric sequence for optimal alignmentand non-identical sequences can be disregarded for comparison purposes).In certain embodiments, the length of a sequence aligned for comparisonpurposes is at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, or 100% of thelength of the reference sequence. The residues at correspondingpositions are then compared. When a position in the first sequence isoccupied by the same residue as the corresponding position in the secondsequence, then the molecules are identical at that position. The percentidentity between the two sequences is a function of the number ofidentical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which needs to be introducedfor optimal alignment of the two sequences. The comparison of sequencesand determination of percent identity between two sequences can beaccomplished using a mathematical algorithm. For example, the percentidentity between two nucleotide sequences can be determined usingmethods such as those described in Computational Molecular Biology,Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing:Informatics and Genome Projects, Smith, D. W., ed., Academic Press, NewYork, 1993; Sequence Analysis in Molecular Biology, von Heinje, G.,Academic Press, 1987; Computer Analysis of Sequence Data, Part I,Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey,1994; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds.,M Stockton Press, New York, 1991; each of which is incorporated hereinby reference. For example, the percent identity between two nucleotidesequences can be determined using the algorithm of Meyers and Miller(CABIOS, 1989, 4:11-17), which has been incorporated into the ALIGNprogram (version 2.0) using a PAM120 weight residue table, a gap lengthpenalty of 12 and a gap penalty of 4. The percent identity between twonucleotide sequences can, alternatively, be determined using the GAPprogram in the GCG software package using an NWSgapdna.CMP matrix.Methods commonly employed to determine percent identity betweensequences include, but are not limited to those disclosed in Carillo,H., and Lipman, D., SIAM J Applied Math., 48:1073 (1988); incorporatedherein by reference. Techniques for determining identity are codified inpublicly available computer programs. Exemplary computer software todetermine homology between two sequences include, but are not limitedto, GCG program package, Devereux, J., et al., Nucleic Acids Research,12(1), 387 (1984)), BLASTP, BLASTN, and FASTA Altschul, S. F. et al., J.Molec. Biol., 215, 403 (1990)).

By “homologs” as it applies to amino acid sequences is meant thecorresponding sequence of other species having substantial identity to asecond sequence of a second species. “Analogs” is meant to includepolypeptide variants which differ by one or more amino acid alterations,e.g., substitutions, additions or deletions of amino acid residues thatstill maintain the properties of the parent polypeptide.

The present invention contemplates several types of glycan-interactingantibodies which are amino acid based including variants andderivatives. These include substitutional, insertional, deletion andcovalent variants and derivatives. As such, included within the scope ofthis invention are glycan-interacting antibody molecules containingsubstitutions, insertions and/or additions, deletions and covalentlymodifications. For example, sequence tags or amino acids, such as one ormore lysines, can be added to the peptide sequences of the invention(e.g., at the N-terminal or C-terminal ends). Sequence tags can be usedfor peptide purification or localization. Lysines can be used toincrease peptide solubility or to allow for biotinylation.Alternatively, amino acid residues located at the carboxy and aminoterminal regions of the amino acid sequence of a peptide or protein mayoptionally be deleted providing for truncated sequences. Certain aminoacids (e.g., C-terminal or N-terminal residues) may alternatively bedeleted depending on the use of the sequence, as for example, expressionof the sequence as part of a larger sequence which is soluble, or linkedto a solid support.

“Substitutional variants” when referring to proteins are those that haveat least one amino acid residue in a native or starting sequence removedand a different amino acid inserted in its place at the same position.The substitutions may be single, where only one amino acid in themolecule has been substituted, or they may be multiple, where two ormore amino acids have been substituted in the same molecule.

As used herein the term “conservative amino acid substitution” refers tothe substitution of an amino acid that is normally present in thesequence with a different amino acid of similar size, charge, orpolarity. Examples of conservative substitutions include thesubstitution of a non-polar (hydrophobic) residue such as isoleucine,valine and leucine for another non-polar residue. Likewise, examples ofconservative substitutions include the substitution of one polar(hydrophilic) residue for another such as between arginine and lysine,between glutamine and asparagine, and between glycine and serine.Additionally, the substitution of a basic residue such as lysine,arginine or histidine for another, or the substitution of one acidicresidue such as aspartic acid or glutamic acid for another acidicresidue are additional examples of conservative substitutions. Examplesof non-conservative substitutions include the substitution of anon-polar (hydrophobic) amino acid residue such as isoleucine, valine,leucine, alanine, methionine for a polar (hydrophilic) residue such ascysteine, glutamine, glutamic acid or lysine and/or a polar residue fora non-polar residue.

“Insertional variants” when referring to proteins are those with one ormore amino acids inserted immediately adjacent to an amino acid at aparticular position in a native or starting sequence. “Immediatelyadjacent” to an amino acid means connected to either the alpha-carboxyor alpha-amino functional group of the amino acid.

“Deletional variants” when referring to proteins, are those with one ormore amino acids in the native or starting amino acid sequence removed.Ordinarily, deletional variants will have one or more amino acidsdeleted in a particular region of the molecule.

As used herein, the term “derivative” is used synonymously with the term“variant” and refers to a molecule that has been modified or changed inany way relative to a reference molecule or starting molecule. In someembodiments, derivatives include native or starting proteins that havebeen modified with an organic proteinaceous or non-proteinaceousderivatizing agent, and post-translational modifications. Covalentmodifications are traditionally introduced by reacting targeted aminoacid residues of the protein with an organic derivatizing agent that iscapable of reacting with selected side-chains or terminal residues, orby harnessing mechanisms of post-translational modifications thatfunction in selected recombinant host cells. The resultant covalentderivatives are useful in programs directed at identifying residuesimportant for biological activity, for immunoassays, or for thepreparation of anti-protein antibodies for immunoaffinity purificationof the recombinant glycoprotein. Such modifications are within theordinary skill in the art and are performed without undueexperimentation.

Certain post-translational modifications are the result of the action ofrecombinant host cells on the expressed polypeptide. Glutaminyl andasparaginyl residues are frequently post-translationally deamidated tothe corresponding glutamyl and aspartyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Either form ofthese residues may be present in the proteins used in accordance withthe present invention.

Other post-translational modifications include hydroxylation of prolineand lysine, phosphorylation of hydroxyl groups of seryl or threonylresidues, methylation of the alpha-amino groups of lysine, arginine, andhistidine side chains (T. E. Creighton, Proteins: Structure andMolecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86(1983)).

Covalent derivatives specifically include fusion molecules in whichproteins of the invention are covalently bonded to a non-proteinaceouspolymer. The non-proteinaceous polymer ordinarily is a hydrophilicsynthetic polymer, i.e. a polymer not otherwise found in nature.However, polymers which exist in nature and are produced by recombinantor in vitro methods are useful, as are polymers which are isolated fromnature. Hydrophilic polyvinyl polymers fall within the scope of thisinvention, e.g. polyvinylalcohol and polyvinylpyrrolidone. Particularlyuseful are polyvinylalkylene ethers such a polyethylene glycol,polypropylene glycol. The proteins may be linked to variousnon-proteinaceous polymers, such as polyethylene glycol, polypropyleneglycol or polyoxyalkylenes, in the manner set forth in U.S. Pat. No.4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

“Features” when referring to proteins are defined as distinct amino acidsequence-based components of a molecule. Features of the proteins of thepresent invention include surface manifestations, local conformationalshape, folds, loops, half-loops, domains, half-domains, sites, terminior any combination thereof.

As used herein when referring to proteins the term “surfacemanifestation” refers to a polypeptide based component of a proteinappearing on an outermost surface.

As used herein when referring to proteins the term “local conformationalshape” means a polypeptide based structural manifestation of a proteinwhich is located within a definable space of the protein.

As used herein when referring to proteins the term “fold” means theresultant conformation of an amino acid sequence upon energyminimization. A fold may occur at the secondary or tertiary level of thefolding process. Examples of secondary level folds include beta sheetsand alpha helices. Examples of tertiary folds include domains andregions formed due to aggregation or separation of energetic forces.Regions formed in this way include hydrophobic and hydrophilic pockets,and the like.

As used herein the term “turn” as it relates to protein conformationmeans a bend which alters the direction of the backbone of a peptide orpolypeptide and may involve one, two, three or more amino acid residues.

As used herein when referring to proteins the term “loop” refers to astructural feature of a peptide or polypeptide which reverses thedirection of the backbone of a peptide or polypeptide and includes fouror more amino acid residues. Oliva et al. have identified at least 5classes of protein loops (J. Mol Biol 266 (4): 814-830; 1997).

As used herein when referring to proteins the term “half-loop” refers toa portion of an identified loop having at least half the number of aminoacid resides as the loop from which it is derived. It is understood thatloops may not always contain an even number of amino acid residues.Therefore, in those cases where a loop contains or is identified toinclude an odd number of amino acids, a half-loop of the odd-numberedloop will include the whole number portion or next whole number portionof the loop (number of amino acids of the loop/2+/-0.5 amino acids). Forexample, a loop identified as a 7 amino acid loop could producehalf-loops of 3 amino acids or 4 amino acids (7/2=3.5+/-0.5 being 3 or4).

As used herein when referring to proteins the term “domain” refers to amotif of a polypeptide having one or more identifiable structural orfunctional characteristics or properties (e.g., binding capacity,serving as a site for protein-protein interactions.

As used herein when referring to proteins the term “half-domain” meansportion of an identified domain having at least half the number of aminoacid resides as the domain from which it is derived. It is understoodthat domains may not always contain an even number of amino acidresidues. Therefore, in those cases where a domain contains or isidentified to include an odd number of amino acids, a half-domain of theodd-numbered domain will include the whole number portion or next wholenumber portion of the domain (number of amino acids of thedomain/2+/-0.5 amino acids). For example, a domain identified as a 7amino acid domain could produce half-domains of 3 amino acids or 4 aminoacids (7/2=3.5+/-0.5 being 3 or 4). It is also understood thatsub-domains may be identified within domains or half-domains, thesesubdomains possessing less than all of the structural or functionalproperties identified in the domains or half domains from which theywere derived. It is also understood that the amino acids of any of thedomain types herein need not be contiguous along the backbone of thepolypeptide (i.e., nonadjacent amino acids may fold structurally toproduce a domain, half-domain or subdomain).

As used herein when referring to proteins the terms “site” as itpertains to amino acid based embodiments is used synonymous with “aminoacid residue” and “amino acid side chain”. A site represents a positionwithin a peptide or polypeptide that may be modified, manipulated,altered, derivatized or varied within the polypeptide based molecules ofthe present invention.

As used herein the terms “termini or terminus” when referring toproteins refers to an extremity of a peptide or polypeptide. Suchextremity is not limited only to the first or final site of the peptideor polypeptide but may include additional amino acids in the terminalregions. The polypeptide based molecules of the present invention may becharacterized as having both an N-terminus (terminated by an amino acidwith a free amino group (NH2)) and a C-terminus (terminated by an aminoacid with a free carboxyl group (COOH)). Proteins of the invention arein some cases made up of multiple polypeptide chains brought together bydisulfide bonds or by non-covalent forces (multimers, oligomers). Thesesorts of proteins will have multiple N- and C-termini. Alternatively,the termini of the polypeptides may be modified such that they begin orend, as the case may be, with a non-polypeptide based moiety such as anorganic conjugate.

Once any of the features have been identified or defined as a componentof a molecule of the invention, any of several manipulations and/ormodifications of these features may be performed by moving, swapping,inverting, deleting, randomizing or duplicating. Furthermore, it isunderstood that manipulation of features may result in the same outcomeas a modification to the molecules of the invention. For example, amanipulation which involved deleting a domain would result in thealteration of the length of a molecule just as modification of a nucleicacid to encode less than a full length molecule would.

Modifications and manipulations can be accomplished by methods known inthe art such as site directed mutagenesis. The resulting modifiedmolecules may then be tested for activity using in vitro or in vivoassays such as those described herein or any other suitable screeningassay known in the art.

Isotopic Variations

The glycan-interacting antibodies of the present invention may containone or more atoms that are isotopes. As used herein, the term “isotope”refers to a chemical element that has one or more additional neutron. Inone embodiment, compounds of the present invention may be deuterated. Asused herein, the term “deuterated” refers to a substance that has hadone or more hydrogen atoms replaced by deuterium isotopes. Deuteriumisotopes are isotopes of hydrogen. The nucleus of hydrogen contains oneproton while deuterium nuclei contain both a proton and a neutron. Theglycan-interacting antibodies may be deuterated in order to change aphysical property of the compound, such as stability, or to allow thecompounds to be used in diagnostic and experimental applications.

Conjugates and Combinations

It is contemplated by the present invention that the glycan-interactingantibodies of the present invention may be complexed, conjugated orcombined with one or more homologous or heterologous molecules. As usedherein, “homologous molecule” means a molecule which is similar in atleast one of structure or function relative to a starting molecule whilea “heterologous molecule” is one that differs in at least one ofstructure or function relative to a starting molecule. Structuralhomologs are therefore molecules which are substantially structurallysimilar. They can be identical. Functional homologs are molecules whichare substantially functionally similar. They can be identical.

Glycan-interacting antibodies of the invention may include conjugates.Such conjugates of the invention may include a naturally occurringsubstance or ligand, such as a protein (e.g., human serum albumin (HSA),low-density lipoprotein (LDL), high-density lipoprotein (HDL), orglobulin); a carbohydrate (e.g., a dextran, pullulan, chitin, chitosan,inulin, cyclodextrin or hyaluronic acid); or a lipid. The ligand mayalso be a recombinant or synthetic molecule, such as a syntheticpolymer, e.g., a synthetic polyamino acid, an oligonucleotide (e.g. anaptamer). Examples of polyamino acids include polyamino acid is apolylysine (PLL), poly L-aspartic acid, poly L-glutamic acid,styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied)copolymer, divinyl ether-maleic anhydride copolymer,N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol(PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllicacid), N-isopropylacrylamide polymers, or polyphosphazine. Example ofpolyamines include: polyethylenimine, polylysine (PLL), spermine,spermidine, polyamine, pseudopeptide-polyamine, peptidomimeticpolyamine, dendrimer polyamine, arginine, amidine, protamine, cationiclipid, cationic porphyrin, quaternary salt of a polyamine, or an alphahelical peptide.

The conjugates can also include targeting groups, e.g., a cell or tissuetargeting agent or group, e.g., a lectin, glycoprotein, lipid orprotein, e.g., an antibody, that binds to a specified cell type such asa kidney cell. A targeting group can be a thyrotropin, melanotropin,lectin, glycoprotein, surfactant protein A, mucin carbohydrate,multivalent lactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-gulucosamine multivalent mannose, multivalent fucose,glycosylated polyaminoacids, multivalent galactose, transferrin,bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, asteroid, bile acid, folate, vitamin B12, biotin, an RGD peptide, an RGDpeptide mimetic or an aptamer.

Targeting groups can be proteins, e.g., glycoproteins, or peptides,e.g., molecules having a specific affinity for a co-ligand, orantibodies e.g., an antibody, that binds to a specified cell type suchas a cancer cell, endothelial cell, or bone cell. Targeting groups mayalso include hormones and hormone receptors. They can also includenon-peptidic species, such as lipids, lectins, carbohydrates, vitamins,cofactors, multivalent lactose, multivalent galactose,N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose,multivalent fucose, or aptamers.

The targeting group can be any ligand that is capable of targeting aspecific receptor. Examples include, without limitation, folate, GalNAc,galactose, mannose, mannose-6P, apatamers, integrin receptor ligands,chemokine receptor ligands, transferrin, biotin, serotonin receptorligands, PSMA, endothelin, GCPII, somatostatin, LDL, and HDL ligands. Inparticular embodiments, the targeting group is an aptamer. The aptamercan be unmodified or have any combination of modifications disclosedherein.

In still other embodiments, glycan-interacting antibodies are covalentlyconjugated to a cell penetrating polypeptide. The cell-penetratingpeptide may also include a signal sequence. The conjugates of theinvention can be designed to have increased stability; increased celltransfection; and/or altered biodistribution (e.g., targeted to specifictissues or cell types).

Conjugating moieties may be added to glycan-interacting antibodies suchthat they allow labeling or flagging targets for clearance. Suchtagging/flagging molecules include, but are not limited to ubiquitin,fluorescent molecules, human influenza hemaglutinin (HA), c-myc [a 10amino acid segment of the human protooncogene myc with sequenceEQKLISEEDL (SEQ ID NO: 265)], histidine (His), flag [a short peptide ofsequence DYKDDDDK (SEQ ID NO: 266)], glutathione S-transferase (GST), V5(a paramyxovirus of simian virus 5 epitope), biotin, avidin,streptavidin, horse radish peroxidase (HRP) and digoxigenin.

In some embodiments, glycan-interacting antibodies may be combined withone another or other molecule in the treatment of a disease orcondition.

Nucleic Acids

The present invention embraces nucleic acid molecules. In someembodiments, nucleic acids encode antibodies of the invention(including, but not limited to antibodies, antibody fragments,intrabodies and chimeric receptor antigens). Such nucleic acid moleculesinclude, without limitation, DNA molecules, RNA molecules,polynucleotides, oligonucleotides, mRNA molecules, vectors, plasmids andother constructs. As used herein, the term “construct” refers to anyrecombinant nucleic acid molecule including, but not limited toplasmids, cosmids, autonomously replicating polynucleotide molecules orlinear or circular single-stranded or double-stranded DNA or RNApolynucleotide molecules. The present invention also embraces cellsprogrammed or generated to express nucleic acid molecules encodingglycan-interacting antibodies. Such cells may be generated throught theuse of transfection, electroporation, viral delivery and the like.Viruses engineered with constructs of the invention may include, but arenot limited to lentiviruses, adenoviruses, adeno-associated viruses andphages. In some cases, nucleic acids of the invention includecodon-optimized nucleic acids. Methods of generating codon-optimizednucleic acids are known in the art and may include, but are not limitedto those described in U.S. Pat. Nos. 5,786,464 and 6,114,148, thecontents of each of which are herein incorporated by reference in theirentirety. In some embodiments, nucleic acid sequence are codon optimizedto improve protein expression or to remove cryptic splice sites.

II. Methods and Uses

Methods of the present disclosure include, but are not limited to,methods of utilizing one or more glycan-interacting antibody fortherapeutic, diagnostic, quantitative, bioprocessing, experimental,and/or investigative purposes. Such glycan-interacting antibodies mayinclude anti-STn antibodies.

Therapeutics Cancer-Related Applications

Aberrant glycosylation is a hallmark of cancer cell transformation.Multiple aberrant glycosylation forms have been described in humancancers, identifying specific tumor-associated carbohydrate antigens(TACAs) as a class of cell surface molecules suitable for specific tumortargeting (Cheever, M.A. et al., Clin Cancer Res. 2009 Sep1;15(17):5323-37). TACA antigen expression has been found in epithelialcancers including, but not limited to, breast, colon, lung, bladder,cervical, ovarian, stomach, prostate, and liver. TACA antigen expressionhas been found in embryonal cancers including, but not limited to, yolksac tumors and seminomas. In addition, TACA antigen expression has beenfound in many melanomas, carcinomas, and leukemias of various tissues(Heimburg-Molinaro et al., Vaccine. 2011 Nov 8: 29(48):8802-8826).Antibodies of the present invention that target one or more TACA arereferred to herein as “anti-TACA antibodies.”

MUC1 is a key cell surface glycoprotein that is normally extensivelyglycosylated but is underglycosylated in tumor cells. Sparseglycosylation of MUC1 leads to exposure of immunogenic antigens. Thesemay be along the MUC1 core peptide sequence or along core carbohydrateresidues. These TACAs include, but are not limited toN-acetylgalactosamine (Tn), sialyl(α2,6)N-acetylgalactosamine (STn) andgalactose(β1-3)N-acetylgalactosamine (also known as Thomsen-Friedenreichantigen or TF). It has been estimated that about 80% of all carcinomasexpress Tn among the core carbohydrates of MUC1 with STn being stronglyexpressed on human carcinoma cells and linked to cancer progression andmetastasis. With few exceptions, Tn and STn are not expressed in normalhealthy tissues. Sialic acid forms a prominent epitope on STn. Theinvention takes advantage of the fact that aberrant Neu5Gc-STn (GcSTn)glycan expression appears to be highly specific to various carcinomas.

In the case of MUC1, Neu5Gc incorporation into STn yields atumor-specific target, a site that is an attractive target forantibody-based therapies to treat tumor tissue. In some embodiments ofthe present invention, glycan-interacting antibodies target MUC1expressing cancer cells that include Neu5Gc. To date, Neu5Gc has beendetected in glycoconjugates from a number of human cancer tissuesincluding, but not limited to colon cancer, retinoblastoma tissue,melanoma, breast cancer and yolk sac tumor tissue. In some embodimentsof the present invention, methods are contemplated forglycan-interacting antibody treatment of these forms of cancer as wellas other forms of cancer, not specifically listed here, characterized bythe presence of cancer cells that include Neu5Gc.

Additional antigens that include glycans have been identified that areexpressed in correlation with cancer (Heimburg-Molinaro, J. et al.,Cancer vaccines and carbohydrate epitopes. Vaccine. 2011 Nov8;29(48):8802-26). These tumor-associated carbohydrate antigens include,but are not limited to blood group Lewis related antigens [including,but not limited to Lewis^(Y) (Le^(Y)), Lewis^(X) (Le^(X)), SialylLewis^(X) (SLe^(X)) and Sialyl Lewis^(A) (SLe^(A))],glycosphingolipid-related antigens [including, but not limited to GloboH, stage-specific embryonic antigen-3 (SSEA-3) and glycosphingolipidsthat include sialic acid], ganglioside-related antigens [including, butnot limited to gangliosides GD2, GD3, GM2, fucosyl GM1 and Neu5GcGM3]and polysialic acid-related antigens.

In some embodiments, therapeutics of the present invention may bedirected toward Lewis blood group antigens. Lewis blood group antigensinclude a fucose residue linked to GlcNAc by an α1-3 linkage or an α1-4linkage. They may be found on both glycolipids and glycoproteins. Lewisblood group antigens may be found in the body fluid of individuals thatare secretors of these antigens. Their appearance on red cells is due toabsorption of Lewis antigens from the serum by the red cells.

In some embodiments, therapeutics of the present invention may bedirected toward Le^(Y). Le^(Y) (also known as CD174) is made up ofGalβ1,4GlcNAC and includes α1,2-as well as α1,3-linked fucose residuesyielding the Fucα(1,2)Galβ(1,4)Fucα(1,3)GlcNAc epitope. It issynthesized from the H antigen by α1,3 fucosyltransferases which attachthe α1,3 fucose to the GlcNAc residue of the parent chain. Le^(Y) may beexpressed in a variety of cancers including, but not limited to ovarian,breast, prostate, colon, lung and epithelial. Due to its low expressionlevel in normal tissues and elevated expression level in many cancers,the Le^(Y) antigen is an attractive target for therapeutic antibodies.

In some embodiments, therapeutics of the present invention may bedirected toward Le^(X). Le^(X) includes the epitopeGalβ1-4(Fucα1-3)GlcNAcβ-R. It is also known as CD15 and stage-specificembryonic antigen-1 (SSEA-1). This antigen was first recognized as beingimmunoreactive with sera taken from a mouse subjected to immunizationwith F9 teratocarcinoma cells. Le^(X) was also found to correlate withembryonic development at specific stages. It is also expressed in avariety of tissues both in the presence and absence of cancer, but canalso be found in breast and ovarian cancers where it is only expressedby cancerous cells.

In some embodiments, therapeutics of the present invention may bedirected toward SLe^(A) and/or SLe^(X). SLe^(A) and SLe^(X) include thestructures Neu5Acα2-3Galβ1-3(Fucα1-4)GlcNAcβ-R andNeu5Acα2-3Galβ1-4(Fucα1-3)GlcNAcβ-R respectively. Their expression isupregulated in cancer cells. The presence of these antigens in serumcorrelates with malignancy and poor prognosis. SLe^(X) is mostly foundas a mucin terminal epitope. It is expressed in a number of differentcancers including breast, ovarian, melanoma, colon, liver, lung andprostate. In some embodiments of the present invention, SLe^(A) andSLe^(X) targets include Neu5Gc (referred to herein as GcSLe^(A) andGcSLe^(X), respectively).

In some embodiments, therapeutics of the present invention may bedirected toward glycolipids and/or epitopes present on glycolipids,including, but not limited to glycosphingolipids. Glycosphingolipidsinclude the lipid ceramide linked to a glycan by the ceramide hydroxylgroup. On the cell membrane, glycosphingolipids form clusters referredto as “lipid rafts”.

In some embodiments, therapeutics of the present invention may bedirected toward Globo H. Globo H is a cancer-related glycosphingolipidfirst identified in breast cancer cells. The glycan portion of Globo Hincludes Fucα(1-2)Galβ(1-3)GalNAcβ(1-3)Galα(1-4)Galβ(1-4)Glcβ(1).Although found in a number of normal epithelial tissues, Globo H hasbeen identified in association with many tumor tissues including, butnot limited to, small cell lung, breast, prostate, lung, pancreatic,gastric, ovarian and endometrial tumors.

In some embodiments, therapeutics of the present invention may bedirected toward gangliosides. Gangliosides are glycosphingolipids thatinclude one or more sialic acid. According to ganglioside nomenclature,G is used as an abbreviation for ganglioside. This abbreviation isfollowed by the letters M, D, or T referring to the number of sialicacid residues attached (1, 2 or 3 respectively). Finally the numbers 1,2 or 3 are used to refer to the order of the distance each migrates whenanalyzed by thin layer chromatography (wherein 3 travels the greatestdistance, followed by 2, and then 1). Gangliosides are known to beinvolved in cancer-related growth and metastasis and may be expressed onthe cell surface of tumor cells. Gangliosides expressed on tumor cellsmay include, but are not limited to GD2, GD3, GM2 and fucosyl GM1 (alsoreferred to herein as Fuc-GM1). In some embodiments of the presentinvention, glycan-interacting antibodies are directed toward GD3. GD3 isa regulator of cell growth. In some embodiments, GD3-directed antibodiesare used to modulate cell growth and/or angiogenesis. In someembodiments, GD3-directed antibodies are used to modulate cellattachment. In some embodiments of the present invention, glycaninteracting antibodies are directed toward GM2. In some embodiments,GM2-directed antibodies are used to modulate cell to cell contact. Insome embodiments, ganglioside targets of the present invention includeone or more Neu5Gc residue. In some embodiments, such targets mayinclude a GM3 variant having Neu5Gc (referred to herein as GcGM3). Theglycan component of GcGM3 is Neu5Gcα2-3Galβ1-4Glc. GcGM3 is a knowncomponent of tumor cells.

In some embodiments, TACAs targeted by anti-TACA antibodies of thepresent invention may include, but are not limited to any of thoselisted in U.S. Publication Nos. US2013/0236486A1, US2013/0108624A1,US2010/0178292A1, US2010/0104572A1, US2012/0039984A1, US2009/0196916A1,and US2009/0041836A1, the contents of each of which are hereinincorporated by reference in their entirety.

In some embodiments, the present invention provides methods of treatingcancer that include the administration of anti-glycan antibodies taughtherein or the administration of compositions of such antibodies (e.g.,compositions of anti-glycan antibodies having at least one excipient).

In some embodiments, methods of the disclosure include completelyeradicating tumor cells to induce durable initial remission throughadministration of one or more glycan-interacting antibodies. Othermethods include inhibition of tumor resurgence for a period of time, insome cases without excessive toxicity. Such periods of time may be fromabout 1 month to about 18 months, from about 1 year to about 5 years,from about 2 years to about 10 years, or greater than 10 years.

STn in Cancer

The immune system has multiple mechanisms for promoting anti-tumor cellimmune activity including both innate and adaptive immune activity. Asused herein, the term “anti-tumor cell immune activity” refers to anyactivity of the immune system that kills or prevents growth and/orproliferation of tumor cells. In some cases, anti-tumor immune activityincludes recognition and tumor cell killing by natural killer (NK) cellsand phagocytosis by macrophages. Adaptive anti-tumor immune responsesinclude tumor antigen uptake and presentation by antigen presentingcells (APCs,) such as dendritic cells (DCs,) leading to modulation of Tcell anti-tumor activity and/or expansion of B cells with secretion oftumor-specific antibodies. The binding of tumor-specific antibodies totumors can lead to antibody-dependent cellular cytotoxicity (ADCC) andcomplement-dependent cytotoxicity (CDC) mechanisms of tumor cell death.

As used herein, the term “immune-resistant tumor cell” refers to a tumorcell that reduces or evades anti-tumor cell immune activity. Somestudies indicate that the expression of STn (a known TACA) on tumor cellsurfaces or secreted into the tumor cell microenvironment can promotetumor cell evasion of anti-tumor immune activity. As used herein, theterm “tumor cell microenvironment” refers to any area adjacent to orsurrounding a tumor cell. Such areas include, but are not limited toareas between tumor cells, between tumor and non-tumor cells,surrounding fluids and surrounding components of the extracellularmatrix.

Sialylated mucins having STn were demonstrated by Ogata et al to reduceNK cell targeting of tumor cells (Ogata, S. et al., 1992. Canc. Res.52:4741-6, the contents of which are herein incorporated by reference intheir entirety). This study found that the presence of ovine, bovine andporcine submaxillary mucin (OSM, BSM and PSM, respectively) led tonearly one hundred percent inhibition of cytotoxicity (see Table 2 ofOgata et al). Further studies by Jandus et al, demonstrate that sometumor cells can evade NK destruction due to the expression ofsialoglycan ligands that can interact with NK cell siglec receptors,leading to NK inhibition (Jandus, C. et al., 2014, JCI. pii: 65899, thecontents of which are herein incorporated by reference in theirentirety).

Studies by Toda et al., demonstrate that STn may bind CD22 receptors onB cells, leading to decreased signal transduction and reduced B cellactivation (Toda, M. et al., 2008. Biochem Biophys Res Commun.372(1):45-50, the contents of which are herein incorporated by referencein their entirety). Dendritic cells (DCs) can affect adaptive immuneactivity by modulating T cell activity. Studies by Carrascal et al foundthat STn expression by bladder cancer cells induced tolerance in DCs,reducing their ability to induce anti-tumor cell immune activity in Tcells (Carrascal, MA et al., 2014. Mol Oncol. pii:S1574-7891(14)00047-7, the contents of which are herein incorporated byreference in their entirety). These studies revealed that DCs cominginto contact with STn-positive bladder cancer cells displayed atolorigenic expression profile with low expression of CD80, CD86, IL-12and TNF-α. Further, DCs were found to modulate regulatory T cells suchthat the T cells had low expression of IFNγ and high expression ofFoxP3. Other studies by van Vliet and others, indicate that DC surfaceexpression of macrophage galactose-type lectin (MGL) can lead totargeting of those cells to tumor tissues (van Vliet, SJ., 2007.Amsterdam: Vrije Universiteit. p1-232 and van Vliet, SJ. et al., 2008. JImmunol. 181(5):3148-55, Nollau, P. et al., 2013. J Histochem Cytochem.61(3):199-205, the contents of each of which are herein incorporated byreference in their entirety). DCs arriving at tissues due to MGLinteractions may influence T helper (Th) cells in one of three ways. DCscan induce T cell tolerance, T cell immune activity or downregulation ofeffector T cells. MGL has been shown to bind to both AcSTn and GcSTn andthe affinity has been analyzed in depth (Mortezai, N. et al., 2013.Glycobiology. 23(7):844-52, the contents of which are hereinincorporated by reference in their entirety). Interestingly, MUC1expression on tumors has been shown to lead to T cell tolerance,protecting tumor cells from immune eradication.

In some embodiments, glycan-interacting antibodies (including, but notlimited to anti-STn antibodies) of the present invention may be used totreat subjects having one or more tumor cells expressing one or moreTACAs. In some cases, glycan-interacting antibodies (including, but notlimited to anti-STn antibodies) of the invention may be used to increaseanti-tumor cell immune activity toward tumor cells expressing STn. Suchantibodies may increase the adaptive immune response and/or the innateimmune response toward immune-resistant tumor cells. Someglycan-interacting antibodies may be used to increase NK anti-tumor cellactivity. Such glycan-interacting antibodies may, in some cases, blockthe interaction between glycan receptors expressed on NK cells and STnglycans on cancer cells or in surrounding tissues.

In some embodiments, glycan-interacting antibodies (including, but notlimited to anti-STn antibodies) of the invention may be used to increaseB cell anti-tumor cell activity. Such antibodies may reduce theinteraction between CD22 receptors on B cells and STn glycans on cancercells or in surrounding tissues. A study by Sjoberg et al. demonstratesthat 9-O-acetylation of α2,6-linked sialic acids on glycoproteins alsoreduced interaction between B cell CD22 receptors and such glycoproteins(Sjoberg, E.R. et al. 1994. JCB. 126(2): 549-562). Another study by Shiet al. reveals that higher levels of 9-O-acetylated sialic acid residueson murine erythroleukemia cells makes these cells more susceptible tocomplement-mediated lysis (Shi, W-X. et al., 1996. J of Biol Chem.271(49): 31526-32, the contents of which are herein incorporated byreference in their entirety). In some embodiments, anti-STn antibodiesof the invention are capable of selectively binding non-9-O-acetylatedSTn, reducing overall STn binding, but reducing tumor cell growth and/orproliferation. (e.g. through increased B cell anti-tumor activity andincreased complement-mediated tumor cell destruction). In someembodiments, glycan-interacting antibodies (including, but not limitedto anti-STn antibodies) of the invention may be used to increase DCanti-tumor activity. Such antibodies may be used to reduce DC toleranceto tumor cells. Reduced DC tolerance may include increasing DCexpression of CD80, CD86, IL-12 and/or TNF-α. In some cases, DCanti-tumor cell activity may include promotion of T cell anti-tumor cellactivity. Such antibodies may prevent binding between DC MGL and glycansexpressed on or around cancer cells.

A study by Ibrahim et al. suggests that high levels of anti-STnantibodies along with endocrine therapy may increase overall survivaland time to progression (TTP) in women with metastatic breast cancer(Ibrahim, N.K. et al., 2013. 4(7): 577-584, the contents of which areherein incorporated by reference in their entirety). In this study,anti-STn antibody levels were elevated after vaccination with STn linkedto keyhole-limpet Hemocyanin (KLH). In some embodiments, anti-STnantibodies of the invention may be used in combination with endocrinetherapy (e.g. tamoxifen and/or an aromatase inhibitor).

In some embodiments, glycan-interacting antibodies of the invention maybe used to reduce or eliminate cancerous cells and/or cells expressingSTn. Such cells include cells that may be part of a tumor.

In some cases, the present invention provides methods of reducing tumorvolumes by administering anti-glycan antibodies of the invention tosubjects with one or more tumors. Reduction in tumor volumes may bedetermined by comparing tumor volumes in a subject before and aftertreatment, or by comparing tumor volumes between anti-glycanantibody-treated and control treated subjects.

In some cases, anti-glycan antibodies of the invention may beadministered to achieve a desired percent reduction in tumor volume in asubject. This may assessed by determining the volume of one or moretumors (e.g., through the use of calipers or imaging techniques like CTscan) in a subject before and after treatment with an anti-glycanantibody and then calculating the percent reduction in tumor volume fromthe two values. In some embodiments, tumor volume in subjects treatedwith anti-glycan antibodies may be reduced by from about 0.1% to about2%, from about 1% to about 5%, from about 3% to about 12%, from about10% to about 30%, from about 20% to about 50%, from about 40% to about60%, from about 50% to about 75%, from about 60% to about 85%, or fromabout 80% to about 99%. In some cases, tumor volume in subjects treatedwith anti-glycan antibodies may be reduced by at least 1%, by at least5%, by at least 10%, by at least 20%, by at least 40%, by at least 50%,by at least 60%, by at least 80%, by at least 85%, by at least 90%, byat least 95%, by at least 98%, by at least 99%, or by 100%.

In some cases, anti-glycan antibodies of the invention may beadministered to achieve a desired percent tumor growth inhibition(%T/C). %T/C is calculated by determining tumor volumes in treatedsubjects and comparing them to tumor volumes in non-treated orplacebo-treated subjects. In some embodiments, the present inventionprovides methods of reducing tumor volume in a subject by administeringan anti-glycan antibody, wherein the %T/C is from about 0.1% to about1%, from about 0.5% to about 5%, from about 2% to about 20%, from about3% to about 16%, from about 10% to about 30%, from about 20% to about60%, or from about 40% to about 80%. In some cases the %T/C is at least80%. In some cases the %T/C is less than 0.1%.

In some embodiments, antibodies used to reduce tumor volumes in subjectsmay be selected based on their ability to bind cell surface glycans(e.g., STn) and/or their ability to kill cancerous cells. In someinstances, antibodies may be selected based on their half-maximaleffective concentration (EC₅₀) for binding cells having cell surfaceSTn. EC₅₀ values for such antibodies may be determined, e.g., throughflow cytometry analysis with cells having cell surface STn. Suchantibodies may have EC50 values of from about 0.1 nM to about 2 nM, fromabout 0.5 nM to about 5 nM, from about 1 nM to about 10 nM, from about 5nM to about 20 nM, or from about 10 nM to about 30 nM.

In some embodiments, the present invention provides methods of killingcancer cells, such as tumor cells, by administering one or moreantibodies presented herein.

In some embodiments, the present disclosure provides a method ofidentifying a subject in need of anti-STn antibody treatment byisolating cancer cells (including, but not limited to cancer stem cells)and/or obtaining biopsy material from a subject and screening the cancercells and/or biopsy material for STn expression. According to suchmethods, subjects with cancer cells and/or biopsy material expressingSTn are deemed to likely benefit from anti-STn antibody treatment or tobe in need of anti-STn antibody treatment (e.g., treatment with one ormore antibody described herein). In some cases, antibodies describedherein may be used for screening of cancer cells and/or biopsy material.Cancer cells may be screened in vitro by culturing the cancer cells anddetecting STn expression using standard immunological assays (e.g.,ELISA, Western blot, or other standard immunological assays). In somecases, cancer cells may be screened for STn expression using flowcytometry techniques. In other embodiments, cancer cells may be grown inculture and tested for viability after treatment with anti-STnantibodies that are antibody-drug conjugates (ADCs). Such ADCs mayinclude a cytotoxic agent, including, but not limited to those describedherein. Cytotoxic agents may include MMAE. Anti-STn antibodies mayinclude humanized antibodies, including, but not limited to, thosedescribed herein. In other embodiments, cancer cells may be screened byusing the cancer cells to form tumors in mice (e.g., NOD/SCID mice). Thetumors developed in mice may be screened by preparing tissue sectionsfrom such tumors and subjecting the tissue sections toimmunohistochemical analysis using anti-STn antibodies, including, butnot limited to anti-STn antibodies described herein. In some cases, thetumors formed in mice may be assessed for changes in tumor volume aftertreatment of the mice with anti-STn antibodies, including, but notlimited to anti-STn antibodies described herein. Such anti-STnantibodies may include ADCs. ADCs may include one or more cytotoxicagent, including, but not limited to any of those described herein(e.g., MMAE). Subjects with cancer cells that demonstrate STn expressionafter screening may be determined to be in need of anti-STn antibodytreatment.

In some embodiments, the present disclosure provides a method ofidentifying an antibody suitable for treating cancer by isolating cancercells (including, but not limited to cancer stem cells) from a subject,screening the cancer cells for STn expression, and contactingSTn-expressing cancer cells with one or more candidate antibodiesspecific for STn to determine whether any of the one or more candidateantibodies are able to bind the cancer cells. As used herein, the term“candidate antibody” refers to an antibody or one of a group ofantibodies that are being evaluated for one or more purposes. Subjectcancer cells may be screened in vitro by culturing the cancer cells anddetecting STn expression using STn-detecting antibodies with standardimmunological assays (e.g., ELISA, Western blot, or other standardimmunological assays) or using flow cytometry techniques. As usedherein, the term “STn-detecting antibody” refers to an antibody thatbinds STn and that allows for observation of such binding either throughthe presence of an incorporated detectable label or through the use of asecondary antibody having a detectable label. In other embodiments,screening the cancer cells may involve using them to form tumors in mice(e.g., NOD/SCID mice). Screening may be carried out by assessing themouse tumors for expression of STn or for reduction in volume afteradministration of anti-STn antibodies, including, but not limited toADCs.

In some embodiments, the present invention includes methods ofevaluating the suitability of an antibody for treating cancer in asubject by obtaining cancer cells from a subject, using the cancer cellsto form tumors in mice (e.g., NOD/SCID mice), administering an anti-STnantibody to the mice, and measuring changes in tumor volume in the mice,wherein if the tumor volume in the mice is decreased, the anti-STnantibody is determined to be suitable for treating cancer in thesubject. In some cases, the anti-STn antibodies are administeredmultiple times. According to such methods, antibodies may beadministered hourly, daily, weekly, monthly, and/or yearly. In somecases, antibodies are administered weekly for a period of from about 2to about 12 weeks. In some cases, antibodies are administered weekly fora period of at least 12 weeks.

STn expression has been implicated in contributing to the metastaticpotential of cancer cells. According to some methods of the disclosure,glycan-interacting antibodies may be used to reduce metastasis. Suchmethods may include the reduction of metastasis by from about 1% toabout 15%, from about 5% to about 25%, from about 10% to about 50%, fromabout 20% to about 60%, from about 30% to about 70%, from about 40% toabout 80%, from about 50% to about 90%, from about 75% to about 95%, orat least 95%.

Cancer Stem Cells as Therapy Targets

Cancer stem cells or CSCs (also called tumor initiating cells) are asubset of cancer cells within a heterogeneous tumor population thatdrive the initiation, growth, dissemination, and recurrence of primaryand metastatic tumors (Karsten and Goletz, SpringerPlus, 2013, 2, 301),which can occur in varying proportions of the total population dependingon tumor type. CSCs are distinguished from terminally differentiatedcells by their capacity to self-renew and give rise to non-CSC,differentiated progeny (Gupta et al., Nature medicine, 2009, 15,1010-1012). These properties are akin to those of normal stem cells.Such distinctions between normal stem cells and CSCs have importantimplications for therapy.

An increasing number of cell-surface biomarkers have been identifiedthat purport to differentiate CSCs from their non-CSC counterparts(Medema et al., Nature cell biology, 2013, 15, 338-344; Zoller, Cancer,2011, 11, 254-267). These may include,but are not limited to CD44,CD133, CD117, and aldehyde dehydrogenase isoform 1 (ALDH1). Althoughsome of these derive from studies of mouse tumors and human cell lines,others have been validated using primary human tumor samples. One ofthese, the membrane-spanning CD44 glycoprotein, or hyaluronan receptor,which is a well-known constituent of a variety of tumor types, has alsomore recently found acceptance as a bona fide CSC marker in humancancers, and in fact is the one most frequently observed (Lobo et al.,2007, 23, 675-699).

CD44 exists in several variant isoforms generated by alternativesplicing events occurring among the 20 exons and 19 introns of thefull-length CD44 gene (Williams et al, Experimental biology andmedicine, 2013, 238, 324-338). Growing experimental evidence points tothe supporting role of CD44 and its variants in contributing to theinnate metastatic and drug resistant phenotype of CSCs (Negi et al.,Journal of drug targeting,2012, 20, 561-573), in part due to modulationof intracellular signal transduction pathways (Williams et al,Experimental biology and medicine, 2013, 238, 324-338). Additionally,patients with triple negative breast cancer, along with several othercancer types, that display high levels of CD44 cells are known to have apoor prognosis and higher mortality (Negi et al., Journal of drugtargeting,2012, 20, 561-573). These observations support the notion thattargeting CD44 offers a means of treating cancer through inhibition orelimination of CSCs, in addition to mature cancer cells. Indeed,numerous approaches to targeting CD44 have been attempted experimentallywith varying degrees of success. These include a wide range oftechnologies that include the use of conjugated and unconjugatedantibodies, nano-carrier drug systems, and hyaluronan-conjugated drugs(Negi et al., Journal of drug targeting, 2012, 20, 561-573). In severalinstances, however, toxic effects were observed in in vivo studies;these untoward side effects may be attributable to the widespreadoccurrence of CD44 and variants on the membranes of most vertebratecells (Naor et al., Seminars in cancer biology, 2008, 18, 260-267), inaddition to its presence on the surface of the targeted CSCs and maturetumor cells. Targeting CD44 protein, which is a constituent of normalhuman stem cells (Williams et al, Experimental biology and medicine,2013, 238, 324-338), can also harm normal stem cell function(Leth-Larsen et al., Molecular medicine, 2012, 18, 1109-1121). Althougha large body of research points to the desirability of targeting CD44protein on CSCs, as well as on mature tumor cells, the intrinsic problemwith this approach remains the present difficulty in designinginhibitors that will spare normal tissue as well as normal stem cells.

Another well-known tumor antigen with implications to CSC biology is theepithelial mucin MUC1, a membrane tethered glycoprotein that isdifferentially expressed at high levels on the majority ofadenocarcinomas but at low levels or not at all on normal epithelialcells. MUC1 has recently been identified as a CSC biomarker on a varietyof neoplasias including breast (Engelmann et al., Cancer research,2008,68, 2419-2426), and pancreatic cancers, where its expression iscorrelated with high metastasis and poor prognosis. As a constituent ofCSCs, MUC1 has been shown to function in cell adhesion, proliferation,survival, and signaling (Engelmann et al., Cancer research,2008, 68,2419-2426) and may also be co-expressed with CD44 (Leth-Larsen et al.,Molecular medicine,2012, 18, 1109-1121). Immunotherapeutic approachesfor targeting MUC1 in cancer are being pursued using vaccines as well asother approaches, but primarily in the context of mature cancer celltherapy (Julien et al., Biomolecules,2012, 2, 435-466; Acres et al.,Expert review of vaccines,2005, 4, 493-502).

Cancer stem cells have been hypothesized to be generated through theepithelial-to-mesenchymal (EMT) transition (Gupta et al., Naturemedicine, 2009, 15, 1010-1012), and /or reversely themesenchymal-to-epithelial (MET) transition that occurs at the site ofmetastasis (Leth-Larsen et al., Molecular medicine,2012, 18, 1109-1121)(also called CSCs plasticity where non-CSCs can give rise to CSCs). Thisdiscovery further underscores the need to eliminate both CSCs andnon-CSCs in a tumor population.

Recent studies with enriched CSC populations has revealed that thesecells, unlike the bulk of the tumor, are relatively quiescent and arepreferentially resistant to many types of current therapies, includingchemotherapy and radiation (Leth-Larsen et al., Molecular medicine,2012,18, 1109-1121). Thus current therapeutic strategies target non-CSCcomponents of the tumor, leaving CSCs largely unaffected only tore-emerge after appropriate cues to reform recurrent primary tumors atthe initial site or to disseminate to distant sites, colonize, andcreate metastatic disease, the major cause of cancer mortality.

Current understanding of the properties of cancer stem cells clearlyemphasized the need not only to target the bulk of cells present intumors, as is current practice, but also the CSC compartment in order topotentially effect complete cures.

As discussed above, strategies that have been developed based on tumor(including CSCs) associated biomarkers face a challenge that most cancerbiomarkers are also present in normal cells including normal stem cells.A therapy that targets a protein biomarker to eliminate CSCs, may alsotarget normal stem cells, causing elimination of normal cells.

Tumor-Specific Glycans in CSCs

Aberrant forms of glycosylation, including appearance of theThomsen-nouveau (Tn) antigen (GalNAc-O-Ser/Thr), have been described innumerous human cancers, identifying glycans as an entirely novel classof tumor-associated carbohydrate antigens suitable for specific tumortargeting (Rabu et al.,. Future oncology, 2012, 8, 943-960). Theformation of the sialyl derivative of Tn (STn) is mediated by the sialyltransferase ST6GalNAc-I which adds sialic acid in an α2,6 linkage to theTn antigen. The sialylation of STn prevents further sugar additions,thus truncating further glycan extensions (Schultz et al., Cancermetastasis reviews, 2012, 31, 501-518).

While the presence of STn in normal adult human tissues is rare, STnoccurs in various human cancers, including ovarian, bladder, breast,cervical, colon, and lung cancer, among others (Ferreira et al.,Molecular oncology, 2013, 7, 719-731; Kinney et al., Cancer,1997, 80,2240-2249). Further, the presence of STn in tumors is associated withmetastatic disease, poor prognosis, and reduced overall survival(Ferreira et al., Molecular oncology, 2013, 7, 719-731; Kinney et al.,Cancer,1997, 80, 2240-2249); therefore, STn is considered a highlyattractive target for cancer detection and therapy. There are twodistinct forms of sialic acid - Neu5Ac and Neu5Gc - located at theterminal position of STn. The Neu5Ac-sialylated form is predominant inhumans since humans cannot synthesize Neu5Gc due to an inactiveCMP-Neu5Ac hydroxylase (CMAH) gene. However, consumption of Neu5Gc-richfoods leads to foreign Neu5Gc incorporation into human cells, especiallyin carcinomas. Previous studies have shown that solid tumors take up andexpress the Neu5Gc form of sialic acid ( Inoue et al.,Glycobiology,2010, 20, 752-762; Malykh et al., Biochimie, 2001, 83,623-634; Padler-Karavani et al., Cancer research, 2011, 71, 3352-3363).mAbs that bind to both glyco-isoforms of STn that are potential cancertargets: Neu5Ac-STn (AcSTn) and Neu5Gc-STn (GcSTn) (i.e., designated aspan-STn antibodies).

STn accumulation is associated with specific somatic mutations observedrepeatedly in solid tumors and with the inactivation of the gene thatencodes the molecular chaperone Core 1Beta3-Galactosyltransferase-Specific Molecular Chaperone (COSMC), whichis required for the formation of active T-synthase (Ju et al.,Nature,2005, 437, 125). T-synthase competes with ST6GalNAc-I for theGalNAc substrate and therefore when inactivated by mutation results inelevated STn synthesis. Additionally, STn accumulation can also resultfrom increased expression of ST6GalNAc-I, which is often observed(Brockhausen et al., Biological chemistry,2001, 382, 219-232; Ikehara etal., Glycobiology,1999, 9, 1213-1224). De novo expression of STn canmodulate carcinoma cells, change the malignant phenotype, and lead tomore aggressive cell behaviors (Pinho et al., Cancer letters, 2007, 249,157-170). As such, STn is not only an interesting cancer biomarker andtherapeutic target, but interfering with STn function offers theintriguing potential to have significant functional, anti-metastatictherapeutic benefits.

Although it is well-known that glycosylation of cellular glycoproteinsis altered in cancer, it appears that aberrant glycosylation isselective with respect to both the glycoprotein and glycan in question.In fact, in human tumor CSCs only CD44 and MUC1 are major carriers ofthe STn antigen (Cazet et al., Breast cancer research : BCR,2010,12,204;Julien et al., Glycobiology, 2006, 16, 54-64), immediatelysuggesting a selective approach for targeting not only mature tumorcells but also CSCs. Whereas MUC1 is a normal surface constituent ofsome epithelial cells where it serves a barrier function,tumor-associated MUC1 is characterized by hypoglycosylation andincreased sialylation on CSCs in the same fashion as observed in maturecancer cells, with STn appearing as a specific marker for both CSCs andmature tumor cells (Curry et al., Journal of surgical oncology,2013,107, 713-722). The aberrant oligosaccharide profile of MUC1 gives riseto the expression of neomarkers such as sialyl-Le^(a) (used in theCA19-9 test), sialyl-Le^(x), and sialyl-Tn (TAG-72), as well as thecryptic epitopes such as Tn in cancer cells (e.g., CSCs). In addition,because of underglycosylation, the peptide core of the mucin becomesexposed such that epitopes within the core (not accessible within normaltissue-derived MUC1) may serve as potential antigens.

Clinical approaches targeting STn have thus far consisted solely of STnvaccines. The most advanced clinical candidate is Theratope, atherapeutic vaccine consisting of STn coupled to keyhole limpethemocyanin. In in vivo mouse studies Theratope immunization induced apotent antibody response that was shown to mediate a delay in the growthof injected STn-expressing mammary carcinoma cells (Julien et al.,British journal of cancer, 2009, 100, 1746-1751). However, Theratopefailed to meet its primary endpoint in a phase III clinical trial inmetastatic breast cancer. A leading hypothesis for why the Theratopetrial missed its primary endpoint is that the patient population was notevaluated for STn expression prior to enrollment. Since STn expressionin breast cancer is highly heterogeneous between patients, ranging from25%-80% depending on the study and detection method, lack of ability tocorrelate STn expression with response may have masked any benefit fromTheratope. Importantly, a subset of patients receiving hormonal therapyshowed a significant 7.5 month increase in median overall survival whentreated with Theratope compared to hormone therapy alone (Ibrahim etal., Journal of clinical oncology : official journal of the AmericanSociety of Clinical Oncology, 2004, 22, 2547;and Miles et al., Theoncologist,2011, 16, 1092-1100), validating the therapeutic potential oftargeting STn in particular patient populations. Additionally, since theimmune response often varies considerably between vaccinated patients,vaccine approaches lack the ability to control or modulate antibodytiter, resulting in wide ranges of therapeutic antibody exposure amongpatients. Nonetheless, Theratope was well tolerated with minimaltoxicity, demonstrating the safety of targeting STn for cancer therapy.

The growing understanding of the molecular basis of STn expression incancer cells strongly suggests that cells that express STn on any cellsurface protein will also express STn on many (if not all) otherO-glycosylated cell surface proteins, rendering it an excellentwidely-distributed cancer-associated therapeutic target. Thus, STnpositive cancer cell populations may be enriched for CSCs. In addition,recent data demonstrate that abrogation of STn expression renderscancers less aggressive with significant reductions in metastaticbehavior ( Gill et al., Proceedings of the National Academy of Sciencesof the United States of America 2013, 110, E3152-3161).

Anti-STn Antibodies Targeting CSCs as Cancer Treatment

Several anti-STn antibodies have been described in the field, but somedemonstrate low specificity towards the STn antigen or sialylatedisoforms. For example, the commercial B72.3 anti-STn antibody has beenshown to bind not only to STn but also to the Tn antigen (Bapat, S. A.(2010) Human ovarian cancer stem cells. Reproduction 140, 33-41). Theavailability of monoclonal antibodies (mAbs) targeting STn, engineeredto induce antibody-dependent cellular cytotoxicity (ADCC) and/orcomplement-dependent cytotoxicity (CDC), or conjugated with a cytotoxicpayload [e.g. antibody drug conjugate (ADC)], offers the potential of asignificant therapeutic benefit for cancer patients with STn-expressingtumors. In addition, such antibodies would also allow for thedevelopment of a companion diagnostic to pre-select patients most likelyto respond to therapy.

STn is often present on one or more of CSC surface antigens, andtogether they serve to promote the stemness and chemoresistanceproperties associated with CSCs. Thus, anti-STn antibodies offer aCSC-associated cancer targeting agent with the potential not only todirectly kill CSCs via direct engagement and/or ADCC, but also offer aunique opportunity to bind to a wide array of cell-surface proteins andinterfere with their associated functions essential for CSC viability,self-renewal, and replication.

As discuss herein, the rationale and advantages of targeting STn on CSCsmay include: (1) many tumor-specific truncated glycoproteins carry STnin cancer; (2) STn is a unique glycan target expressed preferentially onCD44, MUC1, and potentially other important cell-surface markers, onboth CSCs and mature tumor cells, irrespective of proliferation status,allowing for targeting of both of these tumor components by a singletherapeutic agent.; (3) STn is also a component of CA-125, a biomarkerof ovarian cancer and others; (4) STn is a component of the ovarian CSCmarker CD44. Therefore, the use of pan-STn murine mAbs, targeting anepitope that encompasses both the Neu5Ac and Neu5Gc forms of sialic acidlinked to Tn, will bind to and kill or impair the function of CSCs and,by virtue of the common epitope, non-CSC tumor cells.

In some embodiments, the present invention provides new anti-pan STnmAb(s) for specific elimination of human CSCs as well as mature tumorcells. In one aspect of the present invention, the anti-STn antibodywill target the validated STn glycan itself - not a particularglycopeptide or carrier protein, which should offer the broad potentialof binding to CD44, MUC1, or other STn-glycosylated markers on both CSCand non-CSC tumor populations. In some embodiments, glycan-interactingantibodies of the present disclosure may be used to target stemcell-related proteins that have one or more associated glycans. As usedherein, the term “stem cell-related protein” refers to any protein thatis associated with one or more stem cells. Such proteins may include,but are not limited to, cell surface proteins, markers, intracellularproteins, transcription factors, and proteins involved in cellularsignaling that affect stem cell survival, growth, replication, and/ormaintenance. In some cases, such glycans include STn. Stem cell-relatedproteins may include, but are not limited to, Notch, Hedgehog, CD44,CD117, CD133, and integrin.

Given the exceptional specificity in targeting tumor-associated STn, thepresent invention may spare normal tissues, including normal adult stemcells, thereby allowing for an excellent therapeutic window.

In accordance with the present invention, provided herein is a uniqueimmunotherapeutic solution aimed at eradicating human neoplasias byeliminating both CSCs and mature cancer cells contained within the tumorcompartment. The present invention provides therapies and methodsspecifically targeting tumors, which now include targeting CSCs, andhence expanding the therapeutic window by targeting associatedtumor-specific carbohydrate moieties of these potential targets. Theelimination is specifically conferred through targeting tumor associatedcell-surface sialylated Tn antigen (STn) structures that are uniquelypresent in cancer tissue, including cancer stem cells

Ovarian CSCs

Ovarian cancer is the leading gynecological cancer effecting women inthe U.S. During 2013. It is estimated that 22,240 women will bediagnosed with and 14,030 will die of this disease, making it the fifthleading cause of female-related cancer deaths and the most lethalgynecologic malignancy in the U.S. (Siegel et al., Cancer statistics,2013. CA: a cancer journal for clinicians 63, 11-30). This highmortality can be ascribed to non-symptomatic onset, late-stage initialdiagnosis, aggressiveness of this type of cancer, and a general lack oftherapeutically targetable genetic changes. The current standard of careis tumor debulking followed by taxane and platinum based chemotherapy.While this initial treatment results in ~70% of patients achieving aninitial complete clinical response, a majority of these patients willunfortunately relapse with chemoresistant disease (Foster et al., Cancerletters, 2013, 338, 147-157; and McCann et al., PloS one, 2011,6,e28077). In part, recurrent disease has been attributable, as with othercancer types, to the presence of CSCs within the total tumor population.Indeed, ovarian CSCs have been identified and shown to be resistant tochemo-and radiotherapy (Burgos-Ojeda et al., Cancer letters, 2012, 322,1-7). Thus, again as the case with other forms of cancer, eliminatingCSCs along with mature cells in the tumor population offers the besthope to manage recurrent disease and ideally effect cures.

In some embodiments of the present invention, ovarian CSCs may betargeted for ovarian cancer treatment. Although CD133 is the most widelystudied of putative ovarian CSC markers, it is recognized that CD44, aknown carrier of STn as discussed above, is associated with ovariancancer and is included in the set of markers that identify ovarian CSCs(Zhang et al., Cancer research, 2008, 68, 4311-4320; Foster et al.,Cancer letters, 2013, 338, 147-157; and Zoller, Cancer, 2011, 11,254-267). Further, STn is expressed on the well-known ovarian cancerbiomarker CA-125 (MUC16), as well as on MUC1, where the levels of theseSTn-associated mucins in serum have been used recently as furtherdifferentiators of cancerous versus benign ovarian disease. Elevatedserum levels of STn occur in ~50% of ovarian cancer patients andcorrelate with a lower 5-year survival rate (Kobayashi et al., Journalof clinical oncology : official journal of the American Society ofClinical Oncology, 1991, 9, 983-987; Kobayashi et al., Journal ofclinical oncology : official journal of the American Society of ClinicalOncology,1992, 10, 95-101; and Chen et al., Journal of proteomeresearch, 2013, 12, 1408-1418). Finally, Vathipadiekal et al. in a studyof differential gene expression between human primary ovarian carcinomaCSCs and non-CSC populations found that the expression of STn-generatingsialyl transferase ST6GalNAc-I did not differ among cells from the twocompartments.

In some embodiments, the present invention provides antibodies fortargeting CSCs to prevent control or cure cancer related to CSCs. Suchantibodies may include anti-STn antibodies, including, but not limitedto any of those described (or derived from any of those described) ininternational application number PCT/US14/60079, the contents of whichare herein incorporated by reference in their entirety. Further anti-STnantibodies may include antibody 3F1 (SBH Sciences, Natick, MA) orderivatives thereof, including recombinant antibodies with CDRs from 3F1and/or humanized derivatives.

In some embodiments, antibodies of the invention may be used to targetovarian cancer stem cells that are resistant to other forms oftreatment. Such treatments may include chemotherapy. Chemotherapytreatments may include any of those described herein and may include,but are not limited to treatment with carboplatin and/or paclitaxel.Methods of targeting chemotherapy-resistant ovarian cancer stem cellsmay take advantage of changes in cell surface glycan expression inovarian cancer stem cells occurring after chemotherapy treatment. Insome cases, chemotherapy-resistant ovarian cancer stem cells express STnbefore and/or after chemotherapy treatment. After chemotherapytreatments, some chemotherapy-resistant ovarian cancer stem cells mayproliferate resulting in a population of tumor cells that express one ormore cell surface glycans (e.g., STn) that distinguish these cells fromsurrounding cells. Anti-glycan antibodies, including, but not limited tothose presented herein, may be used to kill such populations of ovariancancer stem cells by targeting these distinguishing glycans. In somecases, anti-STn antibodies may be provided. Such antibodies may include,but are not limited to any of the antibodies described herein. In somecases, such antibodies may have at least one variable domain that ishuman or humanized. In some embodiments, subjects having one or morechemotherapy-resistant ovarian cancer stems cells may be treated withanti-STn antibodies of the invention after treatment with carboplatinand/or paclitaxel.

Colorectal Cancer

Colorectal cancer (CRC) has the 4^(th) largest incidence, and iscurrently the third leading cause of cancer-related death in the US.Currently, 20% of patients are diagnosed with metastatic disease androughly 50% of patients with CRC will eventually develop metastases. Forthose diagnosed with metastatic disease, the 5-year survival rate is13.1%. In patients with metastatic colon cancer (mCRC), there isprecedence for use of therapeutic antibodies (e.g., monoclonalantibodies), such as anti-epidermal growth factor receptor (EGFR)monoclonal antibodies and anti-VEGF monoclonal antibodes.

In some embodiments, glycan-interacting antibodies of the presentdisclosure may be used to treat CRC and/or mCRC. In some cases, suchglycan-interacting antibodies are anti-STn antibodies, including, butnot limited to any of those described herein. Glycan-interactingantibodies used to treat CRC and/or mCRC may be conjugated with acytotoxic agent (e.g., MMAE and MMAF). Glycan-interacting antibodies maybe used in combination with other therapies such as therapies with achemotherapeutic agent (e.g., fluoropyrimidine, oxaliplatin, and/oririnotecan) and/or with a therapeutic antibody (e.g., bevacizumab and/oranti-EGFR).

According to some embodiments, glycan-interacting antibodies used totreat colorectal cancer may be administered at a dose of from about 0.5mg/kg to about 20 mg/kg. For example, antibodies may be administered atdoses of from about 0.5 mg/kg to about 2 mg/kg, from about 1 mg/kg toabout 5 mg/kg, from about 2.5 mg/kg to about 10 mg/kg, or from about 5mg/kg to about 20 mg/kg.

Combined Cancer Therapies

In some embodiments, compounds and compositions of the invention may becombined with one or more additional forms of cancer treatment. In somecases, such additional forms may include chemotherapeutic treatments.Accordingly, some methods of the invention include methods of treatingcancer by administering at least one chemotherapeutic agent to a subjecthaving cancer and administering a glycan-interacting antibody. Suchantibodies may include anti-STn antibodies described herein.

As used herein, the term, “chemotherapy” refers to a form of treatmentusing chemical substances. Such chemical substances are referred toherein as “chemotherapeutic agents.” In the treatment of cancer,chemotherapeutic agents are agents that slow or prohibit theproliferation of cancer cells.

In some embodiments, chemotherapeutic agents of the invention may benucleic acid antagonistic agents. Such agents primarily affectproliferating cells, such as cancer cells, and typically function bydisrupting DNA repair and/or synthesis. In some cases, nucleic acidantagonistic agents are alkylating agents (e.g., bifunctional alkylatorsor monofunctional alkylators). Alkylating agents are reactive compoundsthat may be used to disrupt DNA synthesis in dividing cells. Alklyatingagents of the invention may include, but are not limited to,cyclophosphamide, mechlorethamine, chlorambucil, melphalan, decarbazine,nitrosoureas, and temozolomide.

In other embodiments, nucleic acid antagonistic agents of the inventionmay include anthracyclines. Anthracyclines are bacterial derivedcompounds that disrupt nucleic acid synthesis. Anthracyclines of theinvention may include, but are not limited to daunorubicin, doxorubicin,epirubicin, idarubicin, mitoxantrone, and valrubicin. In someembodiments, anthracyclines may be liposomally encapsulated.

In further embodiments, nucleic acid antagonistic agents may be histonedeacetylase inhibitors and/or topoisomerase inhibitors. These inhibitorsprevent changes in DNA supercoiling that are necessary for DNA synthesisand repair. Inhibitors of topoisomerase I may include, but are notlimited to irinotecan and topotecan. Inhibitors of topoisomerase II mayinclude, but are not limited to etoposide, teniposide, and tafluposide.Histone deacetylase inhibitors may include, but are not limited tovorinostat and romidepsin.

In some embodiments, nucleic acid antagonistic agents of the inventionmay include nucleotide analogs and/or nucleotide precursor analogs.Proliferating cells require nucleotides for incorporation into nucleicacids in resulting daughter cells. Nucleotide analogs may disrupt theformation of such nucleic acids or render them non-functional.Nucleotide analogs of the invention may include, but are not limited toazacitidine, azathioprine, capecitabine, doxifluridine, fluorouracil,gemcitabine, hydroxyurea, mercaptopurine, methotrexate, and tioguanine.In some embodiments, leucovorin as administered along with nucleotideanalogs to enhance their effects and/or reduce harmful side effects.

In some embodiments, nucleic acid antagonistic agents of the inventionare platinum-based agents. These agents disrupt nucleic acids bycross-linking them. Platinum-based agents of the invention may include,but are not limited to oxaliplatin, cisplatin, and carboplatin.

In some cases, chemotherapeutic agents of the invention includecytoskeletal disrupting agents. Actively dividing cells undergo majorcytoskeletal changes that may be disrupted by these compounds.Cytoskeletal disrupting agents of the invention may include, but are notlimited to vinca alkaloids, epothilones, paclitaxel, ABRAXANE®(paclitaxel protein-bound particles for injectable suspension), anddocetaxel.

Although effective at targeting proliferating cancer cells,chemotherapeutic agents often affect some non-cancerous cells as well.Because of this, their administration is typically limited by dose,length of treatment, or area of treatment. Further, becausechemotherapeutic agents primarily affect proliferating cells,non-proliferating cancer stem cells may remain viable after treatmentand capable of reforming cancerous cells. Accordingly, in someembodiments, method of the invention include methods of treating cancerin which at least one chemotherapeutic agent is first administered to asubject having cancer, followed by administration of aglycan-interacting antibody. In some cases, the glycan-interactingantibody is selected to target a specific cell surface glycan associatedwith chemotherapy-resistant cells. As used herein, the term“chemotherapy-resistant” is used to refer to cells that are unaffectedby or that have limited susceptibility to chemotherapy treatment.

Methods of targeting chemotherapy-resistant cells (e.g.,chemotherapy-resistant cancer stem cells) may take advantage of changesin STn expression in these cells occurring after chemotherapy treatment.In some cases, chemotherapy-resistant cells express STn before and/orafter chemotherapy treatment. In some cases, cell surface STn expressionin chemoresistant cells may be increased following chemotherapytreatment [e.g., due to altered expression of factors involved in STnsynthesis (e.g., STnGalNAc I, T-synthase, or Cosmc), decreaseddegredation, or other mechanisms leading to increased cell surface STnexpression]. After chemotherapy treatments, some chemotherapy-resistantcells expressing cell surface STn may proliferate resulting in apopulation of STn-expressing tumor cells that arechemotherapy-resistant. In some embodiments, anti-STn antibodies may beused to target chemotherapy-resistant cells. In some cases, these cellsare cancer stem cells. Accordingly, methods of the invention may includemethods of administering an anti-STn antibody to target STn-expressingchemotherapy-resistant cells present after administration of one or morechemotherapeutic agent.

The identification of cell surface glycans on chemotherapy-resistantcells may be carried out by analyzing chemotherapy-resistant cells afterchemotherapy treatment for the identity of cell surface glycans thatdistinguish these cells from surrounding cells. In some embodiments,such cell surface glycans may include, but are not limited tomucin-related antigens (including, but not limited to Tn, STn andThomsen-Friedenreich antigen), blood group Lewis related antigens[including, but not limited to Lewis^(Y) (Le^(Y)), Lewis^(X) (Le^(X)),Sialyl Lewis^(X) (SLe^(X)) and Sialyl Lewis^(A) (SLe^(A))],glycosphingolipid-related antigens [including, but not limited to GloboH, stage-specific embryonic antigen-3 (SSEA-3) and glycosphingolipidshaving sialic acid], ganglioside-related antigens [including, but notlimited to gangliosides GD2, GD3, GM2, fucosyl GM1 and Neu5GcGM3] andpolysialic acid-related antigens. Many of such antigens are described inInternational Publication No. WO2015054600, the contents of which areherein incorporated by reference in their entirety. Analyses carried outto identify cell surface glycans expressed on cancer stem cellsremaining after chemotherapy may be carried out according to any methodsknown in the art. In some cases, such analyses are carried out byobtaining a tissue sample and assessing the expression of cell surfaceglycans in the tissue sample using one or more immunological assay(e.g., immunohistochemical analysis, ELISA analysis, flow cytometricanalysis, antibody array, or mass spectrometry).

In some embodiments, chemotherapy-resistant cells are analyzed to assessthe expression level of cell surface STn. This may be carried out byobtaining a tissue sample and analyzing the sample for expression ofcell surface STn [for example, using one or more immunological assay(e.g., immunohistochemical analysis, ELISA analysis, flow cytometricanalysis, antibody array, or mass spectrometry)]. Wherechemotherapy-resistant cells express STn, anti-STn antibodies may beadministered to a subject after administration of chemotherapeuticagents.

In some embodiments, one or more tumors are primed for treatment withone or more glycan-interacting antibodies by contacting the tumors withat least one chemotherapeutic agent. According to such embodiments,priming a tumor for glycan-interacting antibody treatment refers toreducing proliferating cells in a tumor, leaving one or morechemotherapy-resistant tumor cells behind. According to such methods,glycan-interacting antibodies may be used to further reduce tumorvolumes by eliminating chemotherapy-resistant cells that remain aftertreatment with one or more chemotherapeutic agents.

Administration of glycan-interacting antibodies after administration ofone or more chemotherapeutic agent may be carried out from about 1 dayto about one year after treatment with one or more chemotherapeuticagents (e.g., from about 1 day to about 10 days, from 1 week to about 4weeks, from about 2 weeks to about 10 weeks, from about 1 month to about3 months, from about 2 months to about 6 months, or from about 3 monthsto about 12 months). In some cases, administration of glycan-interactingantibodies may be carried out at least 1 year after treatment with oneor more chemotherapeutic agents.

In some embodiments, multiple rounds of administration with one or morechemotherapeutic agents may be followed by administration ofglycan-interacting antibodies (e.g., 2 rounds, 3 rounds, 4 rounds, 5rounds, 6 rounds, 7 rounds, 8 rounds, 9 rounds, 10 rounds, or at least10 rounds). In some cases, rounds of treatment are repeated until tissueanalyses reveal that cancerous cells and/or chemotherapy-resistant cellsare reduced or eliminated.

The dose of chemotherapeutic agents may be adjusted based on the size ofthe subject receiving treatment. In some embodiments, doses includethose described by Calvo et al. 2014 (Calvo, E. et al., 2014.Chemotherapeutic agents and their uses, dosages, and toxicities. CancerNetwork. p1-12). In some cases, doses are adjusted based on the surfacearea of the subject being treated [typically measured in square meters(m²)]. Chemotherapeutic agents of the invention may be administered atdoses of from about 0.01 mg/m² to about 1 mg/m², from about 0.1 mg/m² toabout 5 mg/m², from about 1 mg/m² to about 20 mg/m², from about 10 mg/m²to about 100 mg/m², from about 50 mg/m² to about 500 mg/m², from about200 mg/m² to about 2000 mg/m², or from about 1000 mg/m² to about 10000mg/m². In some cases, chemotherapeutic agents of the invention areadministered at a dose of at least 10000 mg/m². According to somemethods, chemotherapeutic agents are administered intravenously.

In some embodiments, administration of chemotherapeutic agents includesadministration of carboplatin. According to some methods, carboplatin isadministered at a dose of from about 200 mg/m² to about 400 mg/m². Insome embodiments, administration of chemotherapeutic agents includesadministration of paclitaxel. According to some methods, paclitaxel isadministered at a dose of from about 20 mg/m² to about 300 mg/m².

In some embodiments, glycan-interacting antibodies of the presentdisclosure are administered in combination with anti-angiogenictherapies (e.g., bevacizumab). According to some embodiments, methods oftreating cancer are provided that include identifying a subject in needof cancer treatment, wherein the subject has cancer that is not fullyresponsive to treatment with at least one poly-ADP-ribose polymeraseinhibitor, and administering an anti-STn antibody to the subject. Suchanti-STn antibodies may include any of those known in the art ordescribed herein.

Immune-Related Targets

In some embodiments, glycan-interacting antibodies of the invention maybe immunomodulatory antibodies. As used herein, an immunomodulatoryantibody is an antibody that enhances or suppresses one or more immunefunction or pathway.

Many bacterial glycans are known to include sialic acid. In some cases,such glycans allow bacteria to evade the innate immune system of hosts,including, but not limited to humans. In one example, bacterial glycansinhibit alternate complement pathway activation through factor Hrecognition. In another example, bacterial glycans mask underlyingresidues that may be antigenic. Some bacterial glycans participate incell signaling events through activation of inhibitory sialic acidbinding Ig-like lectins (Siglecs) that dampen the immune response toentities including certain sialylated moieties (Chen, X. et al.,Advances in the biology and chemistry of sialic acids. ACS Chem Biol.2010 Feb 19;5(2):163-76). In some embodiments, glycan-interactingantibodies of the present invention may be used to treat immunecomplications related to bacterial glycans.

Due to the foreign nature of Neu5Gc as described herein, some Neu5Gcglycans are immunogenic resulting in immune related destruction of cellsand other entities where these glycans may be expressed. Such autoimmunedestruction may be pathogenic. In some embodiments, glycan-interactingantibodies may be used to treat patients suffering from autoimmunedisorders related to Neu5Gc glycans.

In some embodiments, immunomodulatory antibodies of the invention may beused to promote or suppress T cell-mediated immunity. Such antibodiesmay interact with one or more glycans present on T cells, T cell-relatedproteins and/or on one or more other cell types that interact with Tcells. Immunomodulatory antibodies that enhance T cell mediated immunitymay be used to stimulate T cell mediated targeting of cancer cells.

In some tumors, infiltration by tumor-associated macrophages (TAMs) maylead to immunosuppression promoting tumor cell viability and growth.This is thought to be due to immunosuppressive cell signaling thatoccurs through interactions between myeloid C-type lectin receptors(CLRs) present on TAMs and tumor-associated mucins (Allavena, P. et al.,Clin Dev Immunol. 2010;2010:547179). In some embodiments, binding ofimmunomodulatory antibodies of the invention to one or moretumor-associated mucin or TACA prevents immunosuppressive cell signalingin TAMs.

Anti-Viral Applications

In some embodiments, glycan-interacting antibodies of the invention maytarget viruses. Viral coat proteins and viral envelopes often includeglycans, referred to herein as viral surface glycans. Such glycans maybe targets of glycan-interacting antibodies. In some embodiments, viralsurface glycans include sialyl-STn. In a further embodiment, viralsurface glycans may include GcSTn. Viruses that may be targeted byglycan-interacting antibodies include, but are not limited to HIV,influenza, rhinovirus, varicella-zoster, rotavirus, herpes (e.g. types 1and 2), hepatitis (e.g. types A, B, C, D and E), yellow fever and humanpapillomavirus.

Other Therapeutic Applications

In some embodiments, glycan-interacting antibodies of the invention mayact to alter or control proteolytic events. In some embodiments,glycan-interacting antibodies of the present invention may beinternalized into cells prior to binding to targets.

Veterinary Applications

It is contemplated that glycan-interacting antibodies of the inventionwill find utility in the area of veterinary care including the care andtreatment of non-human vertebrates. As described herein, the term“non-human vertebrate” includes all vertebrates with the exception ofHomo sapiens, including wild and domesticated species such as companionanimals and livestock. Non-human vertebrates include mammals, such asalpaca, banteng, bison, camel, cat, cattle, deer, dog, donkey, gayal,goat, guinea pig, horse, llama, mule, pig, rabbit, reindeer, sheep waterbuffalo, and yak. Livestock includes domesticated animals raised in anagricultural setting to produce materials such as food, labor, andderived products such as fiber and chemicals. Generally, livestockincludes all mammals, avians and fish having potential agriculturalsignificance. In particular, four-legged slaughter animals includesteers, heifers, cows, calves, bulls, cattle, swine and sheep.

Bioprocessing

In some embodiments of the invention are methods for producingbiological products in host cells by contacting the cells with one ormore glycan-interacting antibody (such as an antibody or fusion protein)capable of modulating gene expression, or altering levels and/or typesof glycans produced wherein such modulation or alteration enhancesproduction of biological products. According to the present invention,bioprocessing methods may be improved by using one or more of theglycan-interacting antibodies of the present invention. They may also beimproved by supplementing, replacing or adding one or moreglycan-interacting antibodies.

Diagnostics

In some embodiments, compounds and compositions of the invention may beused as diagnostics. In some cases, antibodies of the invention may beused to identify, label or stain cells, tissues, organs, etc. expressingtarget antigens. In further embodiments, antibodies of the invention maybe used to identify STn present in tissue sections (i.e., histologicaltissue sections), including tissue known or suspected of havingcancerous cells. Such methods of using antibodies of the invention mayin some cases be used to identify cancerous cells or tumors in tissuesections. Tissue sections may be from any tissue or organ including, butnot limited to breast, colon, pancreatic, ovarian, brain, liver, kidney,spleen, lung, skin, stomach, intestine, esophagous, or bone.

In some embodiments, diagnostic methods of the invention may include theanalysis of one or more cells or tissues using immunohistochemicaltechniques. Such methods may include the use of one or more of any ofthe glycan-interacting antibodies described herein. Immunohistochemicalmethods of the invention may include staining tissue sections todetermine the presence and/or level of one or more glycosylated proteinsor other markers. Tissue sections may be derived from subject tumors(e.g., patient tumors and animal tumors such as animal model tumors).Tissue sections may come from formalin-fixed or unfixed fresh frozentissues. In some case, tissue section come from formalin fixedparaffin-embedded (FFPE) tissues. Glycan-interacting antibodiesdescribed herein may be used as primary antibodies. Primary antibodiesare used to contact tissue sections directly and bind to targetepitopes. Primary antibodies may be directly conjugated with adetectable label or may be detected through the use of a detection agentsuch as a secondary antibody. In some embodiments, primary antibodies ordetection agents include an enzyme that can be used to react with asubstrate to generate a visible product (e.g., precipitate). Suchenzymes may include, but are not limited to horse raddish peroxidase,alkaline phosphatase, beta-galactosidase, and catalase.

Anti-STn antibodies described herein may be used according toimmunohistochemical methods of the present disclosure to detectSTn-glycosylated proteins in tissues or cells. In some cases, theseantibodies are used to detect and/or determine the level of STn in tumortissues. Such tumor tissues may include tumor tissues included in tumormicroarrays. Suitable tumor types include, but are not limitd to breast,colon, ovarian, pancreatic, skin, intestinal, lung, and brain tumors.Levels of anti-STn antibodies used in immunohistochemical stainingtechniques may be varied to increase visible staining or to decreasebackground levels of staining. In some embodiments, antibodyconcentrations of from about 0.01 µg/ml to about 50 µg/ml are used. Forexample, antibody concentrations of from about 0.01 µg/ml to about 1µg/ml, from about 0.05 µg/ml to about 5 µg/ml, from about 0.1 µg/ml toabout 3 µg/ml, from about 1 µg/ml to about 10 µg/ml, from about 2 µg/mlto about 20 µg/ml, from about 3 µg/ml to about 25 µg/ml, from about 4µg/ml to about 30 µg/ml, or from about 5 µg/ml to about 50 µg/ml may beused.

In some embodiments, diagnostic methods of the invention include methodsof generating an STn-linked glycoprotein profile. As used herein theterm “STn-linked glycoprotein profile” refers to a set of informationindicating the level and/or identity of STn-linked glycoproteins in asample or subject. Methods of generating an STn-linked glycoproteinprofile may be carried out on a sample obtained from a subject. Suchsamples may be biological samples including, but not limited to, any ofthose described herein. Biological samples may be cellular samples. Insome cases, cellular samples may include at least one tumor cell. Insome embodiments, tumor cell samples may include BRCA1 mutant ornon-BRCA1 mutant tumor cells.

Glycoproteins included in STn-linked glycoprotein profiles may include,but are not limited to, cancer cell markers, stem cell markers, cancerstem cell markers, and stem cell-related proteins. In some embodiments,glycoproteins identified and/or quantitated as part of a STn-linkedglycoprotein profile may include, but are not limited to CD44, CD133,CD117, integrin, Notch, and Hedgehog.

Levels and/or identities of STn-linked glycoproteins in STn-linkedglycoprotein profiles may be determined according to any methods knownin the art for identifying proteins and/or quantitating protein levels.In some embodiments, such methods may include, but are not limited tomass spectrometry, array analysis (e.g., antibody array or proteinarray), Western blotting, flow cytometry, immunoprecipitation, andELISA. STn-linked glycoproteins may in some cases be immunoprecipitatedfrom a sample prior to analysis. Such immunoprecipitation may be carriedout using an anti-STn antibody. Anti-STn antibodies used forimmunoprecipitation of STn-linked glyocproteins may include any of thoseknown in the art or described herein. In some embodiments,STn-glycoproteins are immunoprecipitated from biological samples usingan anti-STn antibody and then identified and/or quantitated using massspectrometry.

In some embodiments, cancer treatments are informed by STn-linkedglycoprotein profile information. Accordingly, the present disclosureprovides methods of treating cancer that include obtaining a sample froma subject in need of cancer treatment, generating an STn-linkedglycoprotein profile from the sample, selecting a glycan-interactingantibody that binds to an STn-glycosylated protein from the STn-linkedglycoprotein profile, and administering the glycan-interacting antibodyto the subject. Glycan-interacting antibodies administered according tosuch methods may include one or more CDRs or variable domains taughtherein.

In some embodiments, methods of the present disclosure may be used ascompanion diagnostics. As used herein, the term “companion diagnostic”refers to an assay, the results of which aid in the diagnosis ortreatment of subjects. Companion diagnostics may be useful forstratifying patient disease, disorder or condition severity levels,allowing for modulation of treatment regimen and dose to reduce costs,shorten the duration of clinical trial, increase safety and/or increaseeffectiveness. Companion diagnostics may be used to predict thedevelopment of a disease, disorder or condition and aid in theprescription of preventative therapies. Some companion diagnostics maybe used to select subjects for one or more clinical trials. In somecases, companion diagnostic assays may go hand-in-hand with a specifictreatment to facilitate treatment optimization.

In some embodiments, methods of the present disclosure may be useful ascompanion diagnostics for diseases, disorders and/or conditions relatedto cancer. Some companion diagnostics of the present invention may beuseful for predicting and/or determining the severity of one or moreforms of cancer. Some companion diagnostics of the present invention maybe used to stratify subjects by risk of developing one or more forms ofcancer. Some companion diagnostics of the present invention may be usedto facilitate and expedite drug development for cancer therapeutics.

STn Expression-Modified Cells

In some embodiments, the present disclosure provides modified cellshaving altered STn levels. Such cells may may be used for variouspurposes (e.g., experimental, therapeutic, antibody testing etc.). Insome cases, methods of the present disclosure include methods ofenhancing the expression of ST6GalNAc I in one or more cells or tissues.This may result in the generation of one or more cells having increasedexpression of cellular STn (e.g., surface-expressed STn). Expression ofST6GalNAc I may be enhanced, for example, by introducing one or morevectors carrying a ST6GalNAc I expression construct. Such expressionconstructs may be designed with the natural ST6GalNAc I promoter or witha promoter to enhance gene expression. Promoters configured forenhancement of gene expression may have constitutively or overly activepromoter elements. In some cases, promoters may be configured forinducible gene expression. Such promoters may become active or haveelevated activity when contacted with factors that activate inducibleelements of the promoter. STn expression constructs may includehST6GalNAc I pRc-CMV as described in Julien, S. et al., 2001. GlycoconjJ, 18: 883-93, the contents of which are herein incorporated byreference in their entirety. In some embodiments, expression constructsmay encode other factors involved in STn synthesis and/or expression.Such factors may include, but are not limited to, T-synthase, and Core 1Beta3-Galactosyltransferase-Specific Molecular Chaperone (COSMC). Insome embodiments, cells with minimal STn expression are converted toSTn-expressing cells. Such cells may include, but are not limited to,SKOV3 cells, BRCA1 mutant cells, and non-mutant BRCA1 cells.

Also provided are modified cells having decreased STn expressionrelative to unmodified cells. Accordingly, methods of the presentdisclosure include methods of repressing STn expression. Such methodsmay include reducing ST6GalNAc I expression. In some embodiments, suchmethods may include the administration of one or more nucleic acidmolecules that repress ST6GalNAc I expression. Such nucleic acidmolecules may include, but are not limited to inhibitory RNA (e.g., RNAior silencer siRNA). In some embodiments, other factors involved in STnsynthesis and/or expression may be reduced. Such factors may include,but are not limited to T-synthase and COSMC. In some embodiments, cellsnaturally expressing STn are converted to STn-deficient cells. Suchcells may include, but are not limited to, OVCAR3 cells and OVCAR4cells.

III. Pharmaceutical Compositions

In some embodiments, the present disclosure includes pharmaceuticalcompositions. Such pharmaceutical compositions may include antibodies ofthe present disclosure and/or fragments, peptides, or proteins derivedfrom such antibodies. Pharmaceutical compositions may be characterizedby one or more of bioavailability, therapeutic window and/or volume ofdistribution.

Bioavailability

Glycan-interacting antibodies, when formulated into a composition with adelivery/formulation agent or vehicle as described herein, can exhibitan increase in bioavailability as compared to a composition lacking adelivery agent as described herein. As used herein, the term“bioavailability” refers to the systemic availability of a given amountof glycan-interacting antibodies administered to a mammal.Bioavailability can be assessed by measuring the area under the curve(AUC) or the maximum serum or plasma concentration (C_(max)) of theunchanged form of a compound following administration of the compound toa mammal. AUC is a determination of the area under the curve plottingthe serum or plasma concentration of a compound along the ordinate(Y-axis) against time along the abscissa (X-axis). Generally, the AUCfor a particular compound can be calculated using methods known to thoseof ordinary skill in the art and as described in G. S. Banker, ModernPharmaceutics, Drugs and the Pharmaceutical Sciences, v. 72, MarcelDekker, New York, Inc., 1996, herein incorporated by reference.

The C_(max) value is the maximum concentration of the compound achievedin the serum or plasma of a mammal following administration of thecompound to the mammal. The C_(max) value of a particular compound canbe measured using methods known to those of ordinary skill in the art.The phrases “increasing bioavailability” or “improving thepharmacokinetics,” as used herein mean that the systemic availability ofa glycan-interacting antibody, measured as AUC, C_(max), or C_(min) in amammal is greater, when co-administered with a delivery agent asdescribed herein, than when such co-administration does not take place.In some embodiments, the bioavailability of the glycan-interactingantibody can increase by at least about 2%, at least about 5%, at leastabout 10%, at least about 15%, at least about 20%, at least about 25%,at least about 30%, at least about 35%, at least about 40%, at leastabout 45%, at least about 50%, at least about 55%, at least about 60%,at least about 65%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,or about 100%.

Therapeutic Window

Glycan-interacting antibodies, when formulated into a composition with adelivery agent as described herein, can exhibit an increase in thetherapeutic window of the administered glycan-interacting antibodycomposition as compared to the therapeutic window of the administeredglycan-interacting antibody composition lacking a delivery agent asdescribed herein. As used herein “therapeutic window” refers to therange of plasma concentrations, or the range of levels oftherapeutically active substance at the site of action, with a highprobability of eliciting a therapeutic effect. In some embodiments, thetherapeutic window of the glycan-interacting antibody whenco-administered with a delivery agent as described herein can increaseby at least about 2%, at least about 5%, at least about 10%, at leastabout 15%, at least about 20%, at least about 25%, at least about 30%,at least about 35%, at least about 40%, at least about 45%, at leastabout 50%, at least about 55%, at least about 60%, at least about 65%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, or about 100%.

In some embodiments, glycan-interacting antibodies are detectable insubject samples for at least 1 days, at least 2 days, at least 5 days,at least 10 days, at least 14 days, at least 1 month, at least 2 months,at least 6 months, or at least a year after administration. Whereantibodies are conjugated with cytotoxic agents (e.g., MMAE), the drugto antibody ratio (DAR) may remain stable. In some cases, the DAR maychange by less than 1%, by less than 5%, by less than 10%, by less than20%, by less than 30%, by less than 40%, by less than 50%, by less than60%, or by less than 75% over a given period of time (e.g., the periodof time in which antibody levels are detectable in subject samples).

Volume of Distribution

Glycan-interacting antibodies, when formulated into a composition with adelivery agent as described herein, can exhibit an improved volume ofdistribution (V_(dist)), e.g., reduced or targeted, relative to acomposition lacking a delivery agent as described herein. The volume ofdistribution (V_(dist)) relates the amount of the drug in the body tothe concentration of the drug in the blood or plasma. As used herein,the term “volume of distribution” refers to the fluid volume that wouldbe required to contain the total amount of the drug in the body at thesame concentration as in the blood or plasma: V_(dist) equals the amountof drug in the body/concentration of drug in blood or plasma. Forexample, for a 10 mg dose and a plasma concentration of 10 mg/L, thevolume of distribution would be 1 liter. The volume of distributionreflects the extent to which the drug is present in the extravasculartissue. A large volume of distribution reflects the tendency of acompound to bind to the tissue components compared with plasma proteinbinding. In a clinical setting, V_(dist) can be used to determine aloading dose to achieve a steady state concentration. In someembodiments, the volume of distribution of the glycan-interactingantibody when co-administered with a delivery agent as described hereincan decrease at least about 2%, at least about 5%, at least about 10%,at least about 15%, at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 55%, at least about 60%, at leastabout 65%, at least about 70%.

In some embodiments, glycan-interacting antibodies are included incompositions and/or complexes with one or more pharmaceuticallyacceptable excipients. Pharmaceutical compositions may optionallyinclude one or more additional active substances, e.g. therapeuticallyand/or prophylactically active substances. General considerations in theformulation and/or manufacture of pharmaceutical agents may be found,for example, in Remington: The Science and Practice of Pharmacy 21^(st)ed., Lippincott Williams & Wilkins, 2005 (incorporated herein byreference).

In some embodiments, compositions are administered to humans, humanpatients or subjects. For the purposes of the present disclosure, thephrase “active ingredient” generally refers to glycan-interactingantibodies to be delivered as described herein.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for administration to humans, it will be understood by theskilled artisan that such compositions are generally suitable foradministration to any other animal, e.g., to non-human animals, e.g.non-human mammals. Modification of pharmaceutical compositions suitablefor administration to humans in order to render the compositionssuitable for administration to various animals is well understood, andthe ordinarily skilled veterinary pharmacologist can design and/orperform such modification with merely ordinary, if any, experimentation.Subjects to which administration of the pharmaceutical compositions iscontemplated include, but are not limited to, humans and/or otherprimates; mammals, including commercially relevant mammals such ascattle, pigs, horses, sheep, cats, dogs, mice, and/or rats; and/orbirds, including commercially relevant birds such as poultry, chickens,ducks, geese, and/or turkeys.

Formulations of the pharmaceutical compositions described herein may beprepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with an excipient and/orone or more other accessory ingredients, and then, if necessary and/ordesirable, dividing, shaping and/or packaging the product into a desiredsingle- or multi-dose unit.

A pharmaceutical composition in accordance with the invention may beprepared, packaged, and/or sold in bulk, as a single unit dose, and/oras a plurality of single unit doses. As used herein, a “unit dose” isdiscrete amount of the pharmaceutical composition that includes apredetermined amount of the active ingredient. The amount of the activeingredient is generally equal to the dosage of the active ingredientwhich would be administered to a subject and/or a convenient fraction ofsuch a dosage such as, for example, one-half or one-third of such adosage.

Relative amounts of the active ingredient, the pharmaceuticallyacceptable excipient, and/or any additional ingredients in apharmaceutical composition in accordance with the invention will vary,depending upon the identity, size, and/or condition of the subjecttreated and further depending upon the route by which the composition isto be administered. By way of example, the composition may includebetween 0.1% and 100%, e.g., between 0.5 and 50%, between 1-30%, between5-80%, or at least 80% (w/w) active ingredient. In one embodiment,active ingredients are antibodies directed toward cancer cells.

Formulation

Glycan-interacting antibodies of the invention can be formulated usingone or more excipients to: (1) increase stability; (2) increase cellpermeability; (3) permit the sustained or delayed release (e.g., from aformulation of the glycan-interacting antibody); and/or (4) alter thebiodistribution (e.g., target the glycan-interacting antibody tospecific tissues or cell types). In addition to traditional excipientssuch as any and all solvents, dispersion media, diluents, or otherliquid vehicles, dispersion or suspension aids, surface active agents,isotonic agents, thickening or emulsifying agents, preservatives,formulations of the present invention can include, without limitation,liposomes, lipid nanoparticles, polymers, lipoplexes, core-shellnanoparticles, peptides, proteins, cells transfected with theglycan-interacting antibodies (e.g., for transplantation into a subject)and combinations thereof.

Excipients

As used herein, the term “excipient” refers to any substance combinedwith a compound and/or composition of the invention before use. In someembodiments, excipients are inactive and used primarily as a carrier,diluent or vehicle for a compound and/or composition of the presentinvention. Various excipients for formulating pharmaceuticalcompositions and techniques for preparing the composition are known inthe art (see Remington: The Science and Practice of Pharmacy, 21^(st)Edition, A. R. Gennaro, Lippincott, Williams & Wilkins, Baltimore, MD,2006; incorporated herein by reference).

The use of a conventional excipient medium is contemplated within thescope of the present disclosure, except insofar as any conventionalexcipient medium may be incompatible with a substance or itsderivatives, such as by producing any undesirable biological effect orotherwise interacting in a deleterious manner with any othercomponent(s) of the pharmaceutical composition.

Formulations of the pharmaceutical compositions described herein may beprepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofassociating the active ingredient with an excipient and/or one or moreother accessory ingredients.

A pharmaceutical composition in accordance with the present disclosuremay be prepared, packaged, and/or sold in bulk, as a single unit dose,and/or as a plurality of single unit doses.

Relative amounts of the active ingredient, the pharmaceuticallyacceptable excipient, and/or any additional ingredients in apharmaceutical composition in accordance with the present disclosure mayvary, depending upon the identity, size, and/or condition of the subjectbeing treated and further depending upon the route by which thecomposition is to be administered.

In some embodiments, a pharmaceutically acceptable excipient is at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%pure. In some embodiments, an excipient is approved for use in humansand for veterinary use. In some embodiments, an excipient is approved byUnited States Food and Drug Administration. In some embodiments, anexcipient is pharmaceutical grade. In some embodiments, an excipientmeets the standards of the United States Pharmacopoeia (USP), theEuropean Pharmacopoeia (EP), the British Pharmacopoeia, and/or theInternational Pharmacopoeia.

Pharmaceutically acceptable excipients used in the manufacture ofpharmaceutical compositions include, but are not limited to, inertdiluents, dispersing and/or granulating agents, surface active agentsand/or emulsifiers, disintegrating agents, binding agents,preservatives, buffering agents, lubricating agents, and/or oils. Suchexcipients may optionally be included in pharmaceutical compositions.

Exemplary diluents include, but are not limited to, calcium carbonate,sodium carbonate, calcium phosphate, dicalcium phosphate, calciumsulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose,cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol,inositol, sodium chloride, dry starch, cornstarch, powdered sugar, etc.,and/or combinations thereof.

Exemplary granulating and/or dispersing agents include, but are notlimited to, potato starch, corn starch, tapioca starch, sodium starchglycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite,cellulose and wood products, natural sponge, cation-exchange resins,calcium carbonate, silicates, sodium carbonate, cross-linkedpoly(vinylpyrrolidone) (crospovidone), sodium carboxymethyl starch(sodium starch glycolate), carboxymethyl cellulose, cross-linked sodiumcarboxymethyl cellulose (croscarmellose), methylcellulose,pregelatinized starch (starch 1500), microcrystalline starch, waterinsoluble starch, calcium carboxymethyl cellulose, magnesium aluminumsilicate (VEEGUM®), sodium lauryl sulfate, quaternary ammoniumcompounds, etc., and/or combinations thereof.

Exemplary surface active agents and/or emulsifiers include, but are notlimited to, natural emulsifiers (e.g. acacia, agar, alginic acid, sodiumalginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin,egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidalclays (e.g. bentonite [aluminum silicate] and VEEGUM® [magnesiumaluminum silicate]), long chain amino acid derivatives, high molecularweight alcohols (e.g. stearyl alcohol, cetyl alcohol, oleyl alcohol,triacetin monostearate, ethylene glycol distearate, glycerylmonostearate, and propylene glycol monostearate, polyvinyl alcohol),carbomers (e.g. carboxy polymethylene, polyacrylic acid, acrylic acidpolymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives(e.g. carboxymethylcellulose sodium, powdered cellulose, hydroxymethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose,methylcellulose), sorbitan fatty acid esters (e.g. polyoxyethylenesorbitan monolaurate [TWEEN®20], polyoxyethylene sorbitan [TWEENn®60],polyoxyethylene sorbitan monooleate [TWEEN®80], sorbitan monopalmitate[SPAN^(®)40], sorbitan monostearate [Span®60], sorbitan tristearate[Span®65], glyceryl monooleate, sorbitan monooleate [SPAN®80]),polyoxyethylene esters (e.g. polyoxyethylene monostearate [MYRJ®45],polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil,polyoxymethylene stearate, and SOLUTOL®), sucrose fatty acid esters,polyethylene glycol fatty acid esters (e.g. CREMOPHOR®), polyoxyethyleneethers, (e.g. polyoxyethylene lauryl ether [BRIJ®30]),poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamineoleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyllaurate, sodium lauryl sulfate, PLUORINC®F 68, POLOXAMER®188,cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride,docusate sodium, etc. and/or combinations thereof.

Exemplary binding agents include, but are not limited to, starch (e.g.cornstarch and starch paste); gelatin; sugars (e.g. sucrose, glucose,dextrose, dextrin, molasses, lactose, lactitol, mannitol,); natural andsynthetic gums (e.g. acacia, sodium alginate, extract of Irish moss,panwar gum, ghatti gum, mucilage of isapol husks,carboxymethylcellulose, methylcellulose, ethylcellulose,hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, cellulose acetate,poly(vinylpyrrolidone), magnesium aluminum silicate (Veegum®), and larcharabogalactan); alginates; polyethylene oxide; polyethylene glycol;inorganic calcium salts; silicic acid; polymethacrylates; waxes; water;alcohol; etc.; and combinations thereof.

Exemplary preservatives may include, but are not limited to,antioxidants, chelating agents, antimicrobial preservatives, antifungalpreservatives, alcohol preservatives, acidic preservatives, and/or otherpreservatives. Exemplary antioxidants include, but are not limited to,alpha tocopherol, ascorbic acid, acorbyl palmitate, butylatedhydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassiummetabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodiumbisulfite, sodium metabisulfite, and/or sodium sulfite. Exemplarychelating agents include ethylenediaminetetraacetic acid (EDTA), citricacid monohydrate, disodium edetate, dipotassium edetate, edetic acid,fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaricacid, and/or trisodium edetate. Exemplary antimicrobial preservativesinclude, but are not limited to, benzalkonium chloride, benzethoniumchloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride,chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethylalcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol,phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and/orthimerosal. Exemplary antifungal preservatives include, but are notlimited to, butyl paraben, methyl paraben, ethyl paraben, propylparaben, benzoic acid, hydroxybenzoic acid, potassium benzoate,potassium sorbate, sodium benzoate, sodium propionate, and/or sorbicacid. Exemplary alcohol preservatives include, but are not limited to,ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol,chlorobutanol, hydroxybenzoate, and/or phenylethyl alcohol. Exemplaryacidic preservatives include, but are not limited to, vitamin A, vitaminC, vitamin E, beta-carotene, citric acid, acetic acid, dehydroaceticacid, ascorbic acid, sorbic acid, and/or phytic acid. Otherpreservatives include, but are not limited to, tocopherol, tocopherolacetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA),butylated hydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate(SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodiummetabisulfite, potassium sulfite, potassium metabisulfite, GLYDANTPLUS®, PHENONIP®, methylparaben, GERMALL®115, GERMABEN®II, NEOLONE™,KATHON™, and/or EUXYL®.

Exemplary buffering agents include, but are not limited to, citratebuffer solutions, acetate buffer solutions, phosphate buffer solutions,ammonium chloride, calcium carbonate, calcium chloride, calcium citrate,calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconicacid, calcium glycerophosphate, calcium lactate, propanoic acid, calciumlevulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid,tribasic calcium phosphate, calcium hydroxide phosphate, potassiumacetate, potassium chloride, potassium gluconate, potassium mixtures,dibasic potassium phosphate, monobasic potassium phosphate, potassiumphosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride,sodium citrate, sodium lactate, dibasic sodium phosphate, monobasicsodium phosphate, sodium phosphate mixtures, tromethamine, magnesiumhydroxide, aluminum hydroxide, alginic acid, pyrogen-free water,isotonic saline, Ringer’s solution, ethyl alcohol, etc., and/orcombinations thereof.

Exemplary lubricating agents include, but are not limited to, magnesiumstearate, calcium stearate, stearic acid, silica, talc, malt, glycerylbehanate, hydrogenated vegetable oils, polyethylene glycol, sodiumbenzoate, sodium acetate, sodium chloride, leucine, magnesium laurylsulfate, sodium lauryl sulfate, etc., and combinations thereof.

Exemplary oils include, but are not limited to, almond, apricot kernel,avocado, babassu, bergamot, black current seed, borage, cade, camomile,canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, codliver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose,fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop,isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon,litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink,nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel,peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary,safflower, sandalwood, sasquana, savoury, sea buckthorn, sesame, sheabutter, silicone, soybean, sunflower, tea tree, thistle, tsubaki,vetiver, walnut, and wheat germ oils. Exemplary oils include, but arenot limited to, butyl stearate, caprylic triglyceride, caprictriglyceride, cyclomethicone, diethyl sebacate, dimethicone 360,isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol,silicone oil, and/or combinations thereof.

Excipients such as cocoa butter and suppository waxes, coloring agents,coating agents, sweetening, flavoring, and/or perfuming agents can bepresent in the composition, according to the judgment of the formulator.

In some embodiments, anti-glycan antibodies of the invention areformulated with an excipient that includes citrate and/or NaCl. Suchcomposition may include from about 1 mM to about 10 mM, from about 2 mMto about 20 mM, from about 5 mM to about 50 mM, from about 10 mM toabout 100 mM, from about 50 mM to about 200 mM, or from about 100 mM toabout 1,000 mM citrate. Further compositions may include from about 1 mMto about 10 mM, from about 5 mM to about 20 mM, from about 15 mM toabout 50 mM, from about 30 mM to about 60 mM, from about 50 mM to about200 mM, from about 100 mM to about 300 mM, or from about 250 mM to about1000 mM NaCl.

Vehicles Liposomes, Lipoplexes and Lipid Nanoparticles

Glycan-interacting antibodies of the present invention may be formulatedusing one or more liposomes, lipoplexes, or lipid nanoparticles. In oneembodiment, pharmaceutical compositions including glycan-interactingantibodies further include liposomes. Liposomes areartificially-prepared vesicles which may include one or more lipidbilayers and may be used as a delivery vehicle for the administration ofnutrients and pharmaceutical formulations. Liposomes can be of differentsizes such as, but not limited to, a multilamellar vesicle (MLV) whichmay be hundreds of nanometers in diameter and may contain a series ofconcentric bilayers separated by narrow aqueous compartments, a smallunicellular vesicle (SUV) which may be smaller than 50 nm in diameter,and a large unilamellar vesicle (LUV) which may be between 50 and 500 nmin diameter. Liposome design may include, but is not limited to,opsonins or ligands in order to improve the attachment of liposomes tounhealthy tissue or to activate events such as, but not limited to,endocytosis. Liposomes may contain a low or a high pH in order toimprove the delivery of the pharmaceutical formulations.

The formation of liposomes may depend on the physicochemicalcharacteristics such as, but not limited to, the pharmaceuticalformulation entrapped and the liposomal ingredients, the nature of themedium in which the lipid vesicles are dispersed, the effectiveconcentration of the entrapped substance and its potential toxicity, anyadditional processes involved during the application and/or delivery ofthe vesicles, the optimization size, polydispersity and the shelf-lifeof the vesicles for the intended application, and the batch-to-batchreproducibility and possibility of large-scale production of safe andefficient liposomal products.

In one embodiment such formulations may also be constructed orcompositions altered such that they passively or actively are directedto different cell types in vivo.

Formulations can also be selectively targeted through expression ofdifferent ligands on their surface as exemplified by, but not limitedby, folate, transferrin, N-acetylgalactosamine (GalNAc), and antibodytargeted approaches.

Liposomes, lipoplexes, or lipid nanoparticles may be used to improve theefficacy of glycan-interacting antibody function as these formulationsmay be able to increase cell transfection with glycan-interactingantibodies. The liposomes, lipoplexes, or lipid nanoparticles may alsobe used to increase the stability of glycan-interacting antibodies.

Liposomes that are specifically formulated for antibody cargo areprepared according to techniques known in the art, such as described byEppstein et al. (Eppstein, D.A. et al., Biological activity ofliposome-encapsulated murine interferon gamma is mediated by a cellmembrane receptor. Proc Natl Acad Sci U S A. 1985 Jun;82(11):3688-92);Hwang et al. (Hwang, K.J. et al., Hepatic uptake and degradation ofunilamellar sphingomyelin/cholesterol liposomes: a kinetic study. ProcNatl Acad Sci U S A. 1980 Jul;77(7):4030-4); US 4,485,045 and US4,544,545. Production of liposomes with sustained circulation time isalso described in US 5,013,556.

Liposomes that include glycan-interacting antibodies of the presentinvention may be generated using reverse phase evaporation utilizinglipids such as phosphatidylcholine, cholesterol as well asphosphatidylethanolamine that has been polyethylene glycol-derivatized.Filters with defined pore size are used to extrude liposomes of thedesired diameter. In another embodiment, glycan-interacting antibodiesof the present invention can be conjugated to the external surface ofliposomes by disulfide interchange reaction as is described by Martin etal. (Martin, F.J. et al., Irreversible coupling of immunoglobulinfragments to preformed vesicles. An improved method for liposometargeting. J Biol Chem. 1982 Jan 10;257(1):286-8).

Polymers and Nanoparticles

Glycan-interacting antibodies of the invention can be formulated usingnatural and/or synthetic polymers. Non-limiting examples of polymerswhich may be used for delivery include, but are not limited toDMRI/DOPE, poloxamer, chitosan, cyclodextrin, andpoly(lactic-co-glycolic acid) (PLGA) polymers. These may bebiodegradable.

The polymer formulation can permit the sustained or delayed release ofglycan-interacting antibodies (e.g., following intramuscular orsubcutaneous injection). The altered release profile forglycan-interacting antibodies can result in, for example, release of theglycan-interacting antibodies over an extended period of time. Thepolymer formulation may also be used to increase the stability ofglycan-interacting antibodies.

Polymer formulations can also be selectively targeted through expressionof different ligands as exemplified by, but not limited by, folate,transferrin, and N-acetylgalactosamine (GalNAc) (Benoit et al.,Biomacromolecules. 2011 12:2708-2714; Rozema et al., Proc Natl Acad SciU S A. 2007 104:12982-12887; Davis, Mol Pharm. 2009 6:659-668; Davis,Nature 2010 464:1067-1070; herein incorporated by reference in itsentirety).

Glycan-interacting antibodies of the invention can also be formulated asnanoparticles using a combination of polymers, lipids, and/or otherbiodegradable agents, such as, but not limited to, calcium phosphate.Components may be combined in a core-shell, hybrid, and/orlayer-by-layer architecture, to allow for fine-tuning of thenanoparticle so delivery of glycan-interacting antibodies may beenhanced. For glycan-interacting antibodies, systems based onpoly(2-(methacryloyloxy)ethylphosphorylcholine)-block-(2-(diisopropylamino)ethyl methacrylate),(PMPC-PDPA), a pH sensitive diblock copolymer that self-assembles toform nanometer-sized vesicles, also known as polymersomes, atphysiological pH may be used. These polymersomes have been shown tosuccessfully deliver relatively high antibody payloads within livecells. (Massignani, et al, Cellular delivery of antibodies: effectivetargeted subcellular imaging and new therapeutic tool. NatureProceedings, May, 2010).

In one embodiment, a PEG-charge-conversional polymer (Pitella et al.,Biomaterials. 2011 32:3106-3114) may be used to form a nanoparticle todeliver glycan-interacting antibodies of the present invention. ThePEG-charge-conversional polymer may improve upon the PEG-polyanion blockcopolymers by being cleaved into a polycation at acidic pH, thusenhancing endosomal escape.

The use of core-shell nanoparticles has additionally focused on ahigh-throughput approach to synthesize cationic cross-linked nanogelcores and various shells (Siegwart et al., Proc Natl Acad Sci U S A.2011 108:12996-13001). The complexation, delivery, and internalizationof the polymeric nanoparticles can be precisely controlled by alteringthe chemical composition in both the core and shell components of thenanoparticle.

In one embodiment, matrices of poly(ethylene-co-vinyl acetate), are usedto deliver glycan-interacting antibodies of the invention. Such matricesare described in Nature Biotechnology 10, 1446 - 1449 (1992).

Antibody Formulations

Glycan-interacting antibodies of the invention may be formulated forintravenous administration or extravascular administration (Daugherty,et al., Formulation and delivery issues for monoclonal antibodytherapeutics. Adv Drug Deliv Rev. 2006 Aug 7;58(5-6):686-706, U.S. Pat.Publication No.2011/0135570, all of which are incorporated herein intheir entirety). Extravascular administration routes may include, butare not limited to subcutaneous administration, intraperitonealadministration, intracerebral administration, intraocularadministration, intralesional administration, topical administration andintramuscular administration.

Antibody structures may be modified to improve their effectiveness astherapeutics. Improvements may include, but are not limited to improvedthermodynamic stability, reduced Fc receptor binding properties andimproved folding efficiency. Modifications may include, but are notlimited to amino acid substitutions, glycosylation, palmitoylation andprotein conjugation.

Glycan-interacting antibodies may be formulated with antioxidants toreduce antibody oxidation. glycan-interacting antibodies may also beformulated with additives to reduce protein aggregation. Such additivesmay include, but are not limited to albumin, amino acids, sugars, urea,guanidinium chloride, polyalchohols, polymers (such as polyethyleneglycol and dextrans), surfactants (including, but not limited topolysorbate 20 and polysorbate 80) or even other antibodies.

Glycan-interacting antibodies of the present invention may be formulatedto reduce the impact of water on antibody structure and function.Antibody preparations in such formulations may be may be lyophilized.Formulations subject to lyophilization may include carbohydrates orpolyol compounds to protect and stabilize antibody structure. Suchcompounds include, but are not limited to sucrose, trehalose andmannitol.

Glycan-interacting antibodies of the present invention may be formulatedwith polymers. In one embodiment, polymer formulations may containhydrophobic polymers. Such polymers may be microspheres formulated withpolylactide-co-glycolide through a solid-in-oil-in-water encapsulationmethod. Microspheres that include ethylene-vinyl acetate copolymer arealso contemplated for antibody delivery and may be used to extend thetime course of antibody release at the site of delivery. In anotherembodiment, polymers may be aqueous gels. Such gels may, for example,include carboxymethylcellulose. Aqueous gels may also include hyaluronicacid hydrogel. Antibodies may be covalently linked to such gels througha hydrazone linkage that allows for sustained delivery in tissues,including but not limited to the tissues of the central nervous system.

Peptide and Protein Formulations

Glycan-interacting antibodies of the invention may be formulated withpeptides and/or proteins. In one embodiment, peptides such as, but notlimited to, cell penetrating peptides and proteins and peptides thatenable intracellular delivery may be used to deliver pharmaceuticalformulations. A non-limiting example of a cell penetrating peptide whichmay be used with the pharmaceutical formulations of the presentinvention includes a cell-penetrating peptide sequence attached topolycations that facilitates delivery to the intracellular space, e.g.,HIV-derived TAT peptide, penetratins, transportans, or hCT derivedcell-penetrating peptides (see, e.g., Caron et al., Mol. Ther.3(3):310-8 (2001); Langel, Cell-Penetrating Peptides: Processes andApplications (CRC Press, Boca Raton FL, 2002); El-Andaloussi et al.,Curr. Pharm. Des. 11(28):3597-611 (2003); and Deshayes et al., Cell.Mol. Life Sci. 62(16):1839-49 (2005), all of which are incorporatedherein by reference). The compositions can also be formulated to includea cell penetrating agent, e.g., liposomes, which enhance delivery of thecompositions to the intracellular space. Glycan-interacting antibodiesof the invention may be complexed to peptides and/or proteins such as,but not limited to, peptides and/or proteins from Aileron Therapeutics(Cambridge, MA) and Permeon Biologics (Cambridge, MA) in order to enableintracellular delivery (Cronican et al., ACS Chem. Biol. 2010 5:747-752;McNaughton et al., Proc. Natl. Acad. Sci. USA 2009 106:6111-6116;Sawyer, Chem Biol Drug Des. 2009 73:3-6; Verdine and Hilinski, MethodsEnzymol. 2012;503:3-33; all of which are herein incorporated byreference in their entirety).

In one embodiment, cell-penetrating polypeptides may include a firstdomain and a second domain. The first domain may include a superchargedpolypeptide. The second domain may include a protein-binding partner. Asused herein, “protein-binding partner” includes, but are not limited to,antibodies and functional fragments thereof, scaffold proteins, orpeptides. The cell-penetrating polypeptide may further include anintracellular binding partner for the protein-binding partner. Thecell-penetrating polypeptide may be capable of being secreted from acell where glycan-interacting antibodies may be introduced.

In formulations of the present invention, peptides or proteins may beincorporated to increase cell transfection by glycan-interactingantibodies or alter the biodistribution of glycan-interacting antibodies(e.g., by targeting specific tissues or cell types).

Cell Formulations

Cell-based formulations of glycan-interacting antibody compositions ofthe invention may be used to ensure cell transfection (e.g., in thecellular carrier) or alter the biodistribution of the compositions(e.g., by targeting the cell carrier to specific tissues or cell types).

Cell Transfer Methods

A variety of methods are known in the art and are suitable forintroduction of nucleic acids or proteins, such as glycan-interactingantibodies, into a cell, including viral and non-viral mediatedtechniques. Examples of typical non-viral mediated techniques include,but are not limited to, electroporation, calcium phosphate mediatedtransfer, nucleofection, sonoporation, heat shock, magnetofection,liposome mediated transfer, microinjection, microprojectile mediatedtransfer (nanoparticles), cationic polymer mediated transfer(DEAE-dextran, polyethylenimine, polyethylene glycol (PEG) and the like)or cell fusion.

The technique of sonoporation, or cellular sonication, is the use ofsound (e.g., ultrasonic frequencies) for modifying the permeability ofthe cell plasma membrane. Sonoporation methods are known to those in theart and are used to deliver nucleic acids in vivo (Yoon and Park, ExpertOpin Drug Deliv. 2010 7:321-330; Postema and Gilja, Curr PharmBiotechnol. 2007 8:355-361; Newman and Bettinger, Gene Ther. 200714:465-475; all herein incorporated by reference in their entirety).Sonoporation methods are known in the art and are also taught forexample as it relates to bacteria in U.S. Pat. Publication 20100196983and as it relates to other cell types in, for example, U.S. Pat.Publication 20100009424, each of which are incorporated herein byreference in their entirety.

Electroporation techniques are also well known in the art and are usedto deliver nucleic acids in vivo and clinically (Andre et al., Curr GeneTher. 2010 10:267-280; Chiarella et al., Curr Gene Ther. 201010:281-286; Hojman, Curr Gene Ther. 2010 10:128-138; all hereinincorporated by reference in their entirety). In one embodiment,glycan-interacting antibodies may be delivered by electroporation.

Administration and Delivery

The compositions of the present invention may be administered by any ofthe standard methods or routes known in the art.

Glycan-interacting antibodies of the present invention may beadministered by any route which results in a therapeutically effectiveoutcome. These include, but are not limited to enteral, gastroenteral,epidural, oral, transdermal, epidural (peridural), intracerebral (intothe cerebrum), intracerebroventricular (into the cerebral ventricles),epicutaneous (application onto the skin), intradermal, (into the skinitself), subcutaneous (under the skin), nasal administration (throughthe nose), intravenous (into a vein), intraarterial (into an artery),intramuscular (into a muscle), intracardiac (into the heart),intraosseous infusion (into the bone marrow), intrathecal (into thespinal canal), intraperitoneal, (infusion or injection into theperitoneum), intravesical infusion, intravitreal, (through the eye),intracavernous injection, (into the base of the penis), intravaginaladministration, intrauterine, extra-amniotic administration, transdermal(diffusion through the intact skin for systemic distribution),transmucosal (diffusion through a mucous membrane), insufflation(snorting), sublingual, sublabial, enema, eye drops (onto theconjunctiva), or in ear drops. In specific embodiments, compositions maybe administered in a way which allows them cross the blood-brainbarrier, vascular barrier, or other epithelial barrier. Non-limitingroutes of administration for glycan-interacting antibodies of thepresent invention are described below.

Parenteral and Injectable Administration

Liquid dosage forms for oral and parenteral administration include, butare not limited to, pharmaceutically acceptable emulsions,microemulsions, solutions, suspensions, syrups, and/or elixirs. Inaddition to active ingredients, liquid dosage forms may include inertdiluents commonly used in the art such as, for example, water or othersolvents, solubilizing agents and emulsifiers such as ethyl alcohol,isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol,benzyl benzoate, propylene glycol, 1,3-butylene glycol,dimethylformamide, oils (in particular, cottonseed, groundnut, corn,germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfurylalcohol, polyethylene glycols and fatty acid esters of sorbitan, andmixtures thereof. Besides inert diluents, oral compositions can includeadjuvants such as wetting agents, emulsifying and suspending agents,sweetening, flavoring, and/or perfuming agents. In certain embodimentsfor parenteral administration, compositions are mixed with solubilizingagents such as CREMOPHOR®, alcohols, oils, modified oils, glycols,polysorbates, cyclodextrins, polymers, and/or combinations thereof. Inother embodiments, surfactants are included such ashydroxypropylcellulose.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing agents, wetting agents, and/or suspendingagents. Sterile injectable preparations may be sterile injectablesolutions, suspensions, and/or emulsions in nontoxic parenterallyacceptable diluents and/or solvents, for example, as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer’s solution, U.S.P., and isotonic sodiumchloride solution. Sterile, fixed oils are conventionally employed as asolvent or suspending medium. For this purpose any bland fixed oil canbe employed including synthetic mono- or diglycerides. Fatty acids suchas oleic acid can be used in the preparation of injectables. Injectableformulations can be sterilized, for example, by filtration through abacterial-retaining filter, and/or by incorporating sterilizing agentsin the form of sterile solid compositions which can be dissolved ordispersed in sterile water or other sterile injectable medium prior touse.

In order to prolong the effect of an active ingredient, it is oftendesirable to slow the absorption of the active ingredient fromsubcutaneous or intramuscular injection. This may be accomplished by theuse of a liquid suspension of crystalline or amorphous material withpoor water solubility. The rate of absorption of the drug then dependsupon its rate of dissolution which, in turn, may depend upon crystalsize and crystalline form. Alternatively, delayed absorption of aparenterally administered drug form is accomplished by dissolving orsuspending the drug in an oil vehicle. Injectable depot forms are madeby forming microencapsule matrices of the drug in biodegradable polymerssuch as polylactide-polyglycolide. Depending upon the ratio of drug topolymer and the nature of the particular polymer employed, the rate ofdrug release can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissues.

Rectal and Vaginal Administration

Compositions for rectal or vaginal administration are typicallysuppositories which can be prepared by mixing compositions with suitablenon-irritating excipients such as cocoa butter, polyethylene glycol or asuppository wax which are solid at ambient temperature but liquid atbody temperature and therefore melt in the rectum or vaginal cavity andrelease the active ingredient.

Oral Administration

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, an activeingredient is mixed with at least one inert, pharmaceutically acceptableexcipient such as sodium citrate or dicalcium phosphate and/or fillersor extenders (e.g. starches, lactose, sucrose, glucose, mannitol, andsilicic acid), binders (e.g. carboxymethylcellulose, alginates, gelatin,polyvinylpyrrolidinone, sucrose, and acacia), humectants (e.g.glycerol), disintegrating agents (e.g. agar, calcium carbonate, potatoor tapioca starch, alginic acid, certain silicates, and sodiumcarbonate), solution retarding agents (e.g. paraffin), absorptionaccelerators (e.g. quaternary ammonium compounds), wetting agents (e.g.cetyl alcohol and glycerol monostearate), absorbents (e.g. kaolin andbentonite clay), and lubricants (e.g. talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate), andmixtures thereof. In the case of capsules, tablets and pills, the dosageform may include buffering agents.

Topical or Transdermal Administration

As described herein, compositions containing glycan-interactingantibodies of the invention may be formulated for administrationtopically. The skin may be an ideal target site for delivery as it isreadily accessible. Gene expression may be restricted not only to theskin, potentially avoiding nonspecific toxicity, but also to specificlayers and cell types within the skin.

The site of cutaneous expression of the delivered compositions willdepend on the route of nucleic acid delivery. Three routes are commonlyconsidered to deliver glycan-interacting antibodies to the skin: (i)topical application (e.g. for local/regional treatment and/or cosmeticapplications); (ii) intradermal injection (e.g. for local/regionaltreatment and/or cosmetic applications); and (iii) systemic delivery(e.g. for treatment of dermatologic diseases that affect both cutaneousand extracutaneous regions). glycan-interacting antibodies can bedelivered to the skin by several different approaches known in the art.

In one embodiment, the invention provides for a variety of dressings(e.g., wound dressings) or bandages (e.g., adhesive bandages) forconveniently and/or effectively carrying out methods of the presentinvention. Dressings or bandages may include sufficient amounts ofpharmaceutical compositions and/or glycan-interacting antibodiesdescribed herein to allow a user to perform multiple treatments of asubject(s).

In one embodiment, the invention provides for compositions that includeglycan-interacting antibodies to be delivered in more than oneinjection.

Dosage forms for topical and/or transdermal administration of acomposition may include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants and/or patches. Generally, anactive ingredient is admixed under sterile conditions with apharmaceutically acceptable excipient and/or any needed preservativesand/or buffers as may be required.

Additionally, the present invention contemplates the use of transdermalpatches, which often have the added advantage of providing controlleddelivery of a compound to the body. Such dosage forms may be prepared,for example, by dissolving and/or dispensing the compound in the propermedium. Alternatively or additionally, rate may be controlled by eitherproviding a rate controlling membrane and/or by dispersing the compoundin a polymer matrix and/or gel.

Formulations suitable for topical administration include, but are notlimited to, liquid and/or semi liquid preparations such as liniments,lotions, oil in water and/or water in oil emulsions such as creams,ointments and/or pastes, and/or solutions and/or suspensions.

Topically-administrable formulations may, for example, include fromabout 1% to about 10% (w/w) active ingredient, although theconcentration of active ingredient may be as high as the solubilitylimit of the active ingredient in the solvent. Formulations for topicaladministration may further include one or more of the additionalingredients described herein.

Depot Administration

As described herein, in some embodiments, compositions of the presentinvention are formulated in depots for extended release. Generally, aspecific organ or tissue (a “target tissue”) is targeted foradministration.

In some aspects of the invention, glycan-interacting antibodies arespatially retained within or proximal to a target tissue. Provided aremethods of providing compositions to one or more target tissue of amammalian subject by contacting the one or more target tissue (includingone or more target cells) with compositions under conditions such thatthe compositions, in particular glycan-interacting antibody component(s)of the compositions, are substantially retained in the target tissue,meaning that at least 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96,97, 98, 99, 99.9, 99.99 or greater than 99.99% of the composition isretained in the target tissue. Advantageously, retention is determinedby measuring the level of glycan-interacting antibodies present in thecompositions entering the target tissues and/or cells. For example, atleast 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99,99.9, 99.99 or greater than 99.99% of glycan-interacting antibodiesadministered to the subject are present intracellularly at a period oftime following administration. For example, intramuscular injection to amammalian subject may be performed using an aqueous compositionincluding one or more glycan-interacting antibody and a transfectionreagent, and retention of the composition may be determined by measuringthe level of glycan-interacting antibodies present in the muscle cells.

Certain aspects of the invention are directed to methods of providingcompositions to target tissues of mammalian subjects, by contacting thetarget tissues (containing one or more target cells) with compositionsunder conditions such that the compositions are substantially retainedin the target tissue. Compositions contain an effective amount ofglycan-interacting antibodies such that the effect of interest isproduced in at least one target cell. Compositions generally containcell penetration agents and a pharmaceutically acceptable carrier,although “naked” glycan-interacting antibodies (such asglycan-interacting antibodies without cell penetration agents or otheragents) are also contemplated.

In some embodiments, compositions include a plurality of differentglycan-interacting antibodies, where one or more than one of theglycan-interacting antibodies targets a glycan of interest. Optionally,compositions also contain cell penetration agents to assist in theintracellular delivery of compositions. A determination is made of thecomposition dose required to target glycans of interest in a substantialpercentage of cells contained within a predetermined volume of thetarget tissue (generally, without targeting glycans in tissue adjacentto the predetermined volume, or distally to target tissues). Subsequentto this determination, the determined dose may be introduced directlyinto the tissue of the mammalian subject.

In one embodiment, the invention provides for glycan-interactingantibodies to be delivered in more than one injection or by split doseinjections.

Pulmonary Administration

Pharmaceutical compositions may be prepared, packaged, and/or sold informulations suitable for pulmonary administration via the buccalcavity. Such formulations may include dry particles further includingactive ingredients and having a diameter in the range from about 0.5 nmto about 7 nm or from about 1 nm to about 6 nm. Such compositions may besuitably in the form of dry powders for administration using a devicethat includes a dry powder reservoir to which a stream of propellant maybe directed to disperse the powder and/or using a self-propellingsolvent/powder dispensing container such as a device including theactive ingredient dissolved and/or suspended in a low-boiling propellantin a sealed container. Such powders may include particles wherein atleast 98% of the particles by weight have a diameter greater than 0.5 nmand at least 95% of the particles by number have a diameter less than 7nm. Alternatively, at least 95% of the particles by weight have adiameter greater than 1 nm and at least 90% of the particles by numberhave a diameter less than 6 nm. Dry powder compositions may include asolid fine powder diluent such as sugar and are conveniently provided ina unit dose form.

Low boiling propellants generally include liquid propellants having aboiling point of below 65° F. at atmospheric pressure. Generally thepropellant may constitute 50% to 99.9% (w/w) of the composition, andactive ingredient may constitute 0.1% to 20% (w/w) of the composition. Apropellant may further include additional ingredients such as a liquidnonionic and/or solid anionic surfactant and/or a solid diluent (whichmay have a particle size of the same order as particles that include theactive ingredient).

Pharmaceutical compositions formulated for pulmonary delivery mayprovide an active ingredient in the form of droplets of a solutionand/or suspension. Such formulations may be prepared, packaged, and/orsold as aqueous and/or dilute alcoholic solutions and/or suspensions,optionally sterile, that include active ingredient, and may convenientlybe administered using any nebulization and/or atomization device. Suchformulations may further include one or more additional ingredientsincluding, but not limited to, a flavoring agent such as saccharinsodium, a volatile oil, a buffering agent, a surface active agent,and/or a preservative such as methylhydroxybenzoate. Droplets providedby this route of administration may have an average diameter in therange from about 0.1 nm to about 200 nm.

Intranasal, Nasal and Buccal Administration

Formulations described herein as being useful for pulmonary delivery areuseful for intranasal delivery of a pharmaceutical composition. Anotherformulation suitable for intranasal administration is a coarse powdercomprising the active ingredient and having an average particle fromabout 0.2 µm to 500 µm. Such a formulation is administered in the mannerin which snuff is taken, i.e. by rapid inhalation through the nasalpassage from a container of the powder held close to the nose.

Formulations suitable for nasal administration may, for example,comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) ofactive ingredient, and may comprise one or more of the additionalingredients described herein. A pharmaceutical composition may beprepared, packaged, and/or sold in a formulation suitable for buccaladministration. Such formulations may, for example, be in the form oftablets and/or lozenges made using conventional methods, and may, forexample, 0.1% to 20% (w/w) active ingredient, the balance comprising anorally dissolvable and/or degradable composition and, optionally, one ormore of the additional ingredients described herein. Alternately,formulations suitable for buccal administration may comprise a powderand/or an aerosolized and/or atomized solution and/or suspensioncomprising active ingredient. Such powdered, aerosolized, and/oraerosolized formulations, when dispersed, may have an average particleand/or droplet size in the range from about 0.1 nm to about 200 nm, andmay further comprise one or more of any additional ingredients describedherein.

Ophthalmic or Otic Administration

Pharmaceutical compositions may be prepared, packaged, and/or sold in aformulation suitable for ophthalmic or otic administration. Suchformulations may, for example, be in the form of eye or ear dropsincluding, for example, a 0.1/1.0% (w/w) solution and/or suspension ofthe active ingredient in an aqueous or oily liquid excipient. Such dropsmay further comprise buffering agents, salts, and/or one or more otherof any additional ingredients described herein. Otherophthalmically-administrable formulations which are useful include thosewhich comprise the active ingredient in microcrystalline form and/or ina liposomal preparation. Subretinal inserts may also be used as a formof administration.

Payload Administration

Glycan-interacting antibodies described herein may be used in a numberof different scenarios in which delivery of a substance (the “payload”)to a biological target is desired, for example delivery of detectablesubstances for detection of the target, or delivery of a therapeutic ordiagnostic agent. Detection methods can include, but are not limited to,both imaging in vitro and in vivo imaging methods, e.g.,immunohistochemistry, bioluminescence imaging (BLI), Magnetic ResonanceImaging (MRI), positron emission tomography (PET), electron microscopy,X-ray computed tomography, Raman imaging, optical coherence tomography,absorption imaging, thermal imaging, fluorescence reflectance imaging,fluorescence microscopy, fluorescence molecular tomographic imaging,nuclear magnetic resonance imaging, X-ray imaging, ultrasound imaging,photoacoustic imaging, lab assays, or in any situation wheretagging/staining/imaging is required.

Glycan-interacting antibodies can be designed to include both a linkerand a payload in any useful orientation. For example, a linker havingtwo ends is used to attach one end to the payload and the other end tothe glycan-interacting antibody. The glycan-interacting antibodies ofthe invention can include more than one payload as well as a cleavablelinker. In another example, a drug that may be attached toglycan-interacting antibodies via a linker and may be fluorescentlylabeled can be used to track the drug in vivo, e.g. intracellularly.

Other examples include, but are not limited to, the use ofglycan-interacting antibodies in reversible drug delivery into cells.

Glycan-interacting antibodies described herein can be used inintracellular targeting of a payload, e.g., detectable or therapeuticagents, to specific organelles. In addition, glycan-interactingantibodies described herein may be used to deliver therapeutic agents tocells or tissues, e.g., in living animals. For example,glycan-interacting antibodies described herein may be used to deliverchemotherapeutic agents to kill cancer cells. glycan-interactingantibodies attached to therapeutic agents through linkers can facilitatemember permeation allowing the therapeutic agent to travel into a cellto reach an intracellular target.

In some embodiments, the payload may be a therapeutic agent such as acytotoxin, radioactive ion, chemotherapeutic, or other therapeuticagent. A cytotoxin or cytotoxic agent includes any agent that may bedetrimental to cells. Examples include, but are not limited to, taxol,cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,etoposide, teniposide, vincristine, vinblastine, colchicine,doxorubicin, daunorubicin, dihydroxyanthracinedione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids,e.g., maytansinol (see U.S. Pat. No. 5,208,020 incorporated herein inits entirety), rachelmycin (CC-1065, see U.S. Pat. Nos. 5,475,092,5,585,499, and 5,846,545, all of which are incorporated herein byreference), and analogs or homologs thereof. Radioactive ions include,but are not limited to iodine (e.g., iodine 125 or iodine 131),strontium 89, phosphorous, palladium, cesium, iridium, phosphate,cobalt, yttrium 90, samarium 153, and praseodymium. Other therapeuticagents include, but are not limited to, antimetabolites (e.g.,methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine,thiotepa chlorambucil, rachelmycin (CC-1065), melphalan, carmustine(BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine, vinblastine, taxol and maytansinoids). In the caseof anti-STn antibodies of the present invention, tumor killing may beboosted by the conjugation of a toxin to such anti-STn antibodies.

In some embodiments, the payload may be a detectable agent, such asvarious organic small molecules, inorganic compounds, nanoparticles,enzymes or enzyme substrates, fluorescent materials, luminescentmaterials (e.g., luminol), bioluminescent materials (e.g., luciferase,luciferin, and aequorin), chemiluminescent materials, radioactivematerials (e.g., ¹⁸F, ⁶⁷Ga, ^(81m)Kr, ⁸²Rb, ¹¹¹In, ¹²³I, ¹³³Xe, ²⁰¹Tl,¹²⁵I, ³⁵S, ¹⁴C, ³H, or ^(99m)Tc (e.g., as pertechnetate(technetate(VII), TcO₄ ⁻)), and contrast agents (e.g., gold (e.g., goldnanoparticles), gadolinium (e.g., chelated Gd), iron oxides (e.g.,superparamagnetic iron oxide (SPIO), monocrystalline iron oxidenanoparticles (MIONs), and ultrasmall superparamagnetic iron oxide(USPIO)), manganese chelates (e.g., Mn-DPDP), barium sulfate, iodinatedcontrast media (iohexol), microbubbles, or perfluorocarbons). Suchoptically-detectable labels include for example, without limitation,4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid; acridine andderivatives (e.g., acridine and acridine isothiocyanate);5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS);4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate;N-(4-anilino-1-naphthyl)maleimide; anthranilamide; BODIPY; BrilliantYellow; coumarin and derivatives (e.g., coumarin,7-amino-4-methylcoumarin (AMC, Coumarin 120), and7-amino-4-trifluoromethylcoumarin (Coumarin 151)); cyanine dyes;cyanosine; 4′,6-diaminidino-2-phenylindole (DAPI); 5′5″-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red);7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin;diethylenetriamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid;4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid;5-[dimethylamino]-naphthalene-1-sulfonyl chloride (DNS, dansylchloride);4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin andderivatives (e.g., eosin and eosin isothiocyanate); erythrosin andderivatives (e.g., erythrosin B and erythrosin isothiocyanate);ethidium; fluorescein and derivatives (e.g., 5-carboxyfluorescein (FAM),5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),2′,7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein, fluorescein,fluorescein isothiocyanate, X-rhodamine-5-(and-6)-isothiocyanate (QFITCor XRITC), and fluorescamine);2-[2-[3-[[1,3-dihydro-1,1-dimethyl-3-(3-sulfopropyl)-2H-benz[e]indol-2-ylidene]ethylidene]-2-[4-(ethoxycarbonyl)-1-piperazinyl]-1-cyclopenten-1-yl]ethenyl]-1,1-dimethyl-3-(3-sulforpropyl)-1H-benz[e]indoliumhydroxide, inner salt, compound with n,n-diethylethanamine(1:1) (IR144);5-chloro-2-[2-[3-[(5-chloro-3-ethyl-2(3H)-benzothiazol-ylidene)ethylidene]-2-(diphenylamino)-1-cyclopenten-1-yl]ethenyl]-3-ethylbenzothiazolium perchlorate (IR140); Malachite Green isothiocyanate;4-methylumbelliferone orthocresolphthalein; nitrotyrosine;pararosaniline; Phenol Red; B-phycoerythrin; o-phthaldialdehyde; pyreneand derivatives(e.g., pyrene, pyrene butyrate, and succinimidyl1-pyrene); butyrate quantum dots; Reactive Red 4 (CIBACRON™ BrilliantRed 3B-A); rhodamine and derivatives (e.g., 6-carboxy-X-rhodamine (ROX),6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloriderhodarnine (Rhod), rhodamine B, rhodamine 123, rhodamine Xisothiocyanate, sulforhodamine B, sulforhodamine 101, sulfonyl chloridederivative of sulforhodamine 101 (Texas Red),N,N,N′,N′tetramethyl-6-carboxyrhodamine (TAMRA) tetramethyl rhodamine,and tetramethyl rhodamine isothiocyanate (TRITC)); riboflavin; rosolicacid; terbium chelate derivatives; Cyanine-3 (Cy3); Cyanine-5 (Cy5);cyanine-5.5 (Cy5.5), Cyanine-7 (Cy7); IRD 700; IRD 800; Alexa 647; LaJolta Blue; phthalo cyanine; and naphthalo cyanine.

In some embodiments, the detectable agent may be a non-detectableprecursor that becomes detectable upon activation (e.g., fluorogenictetrazine-fluorophore constructs (e.g., tetrazine-BODIPY FL,tetrazine-Oregon Green 488, or tetrazine-BODIPY TMR-X) or enzymeactivatable fluorogenic agents (e.g., PROSENSE® (VisEn Medical))). Invitro assays in which the enzyme labeled compositions can be usedinclude, but are not limited to, enzyme linked immunosorbent assays(ELISAs), immunoprecipitation assays, immunofluorescence, enzymeimmunoassays (EIA), radioimmunoassays (RIA), and Western blot analysis.

Combinations

Glycan-interacting antibodies may be used in combination with one ormore other therapeutic, prophylactic, diagnostic, or imaging agents. By“in combination with,” it is not intended to imply that the agents mustbe administered at the same time and/or formulated for deliverytogether, although these methods of delivery are within the scope of thepresent disclosure. Compositions can be administered concurrently with,prior to, or subsequent to, one or more other desired therapeutics ormedical procedures. In general, each agent will be administered at adose and/or on a time schedule determined for that agent. In someembodiments, the present disclosure encompasses the delivery ofpharmaceutical, prophylactic, diagnostic, and/or imaging compositions incombination with agents that may improve their bioavailability, reduceand/or modify their metabolism, inhibit their excretion, and/or modifytheir distribution within the body.

Dosage

The present disclosure encompasses delivery of glycan-interactingantibodies for any of therapeutic, pharmaceutical, diagnostic or imagingby any appropriate route taking into consideration likely advances inthe sciences of drug delivery. Delivery may be naked or formulated.

Naked Delivery

Glycan-interacting antibodies of the present invention may be deliveredto cells, tissues, organs or organisms in naked form. As used herein in,the term “naked” refers to glycan-interacting antibodies delivered freefrom agents or modifications which promote transfection or permeability.Naked glycan-interacting antibodies may be delivered to cells, tissues,organs and/or organisms using routes of administration known in the artand described herein. Naked delivery may include formulation in a simplebuffer such as saline or PBS.

Formulated Delivery

Glycan-interacting antibodies of the present invention may beformulated, using methods described herein. Formulations may includeglycan-interacting antibodies which may be modified and/or unmodified.Formulations may further include, but are not limited to, cellpenetration agents, pharmaceutically acceptable carriers, deliveryagents, bioerodible or biocompatible polymers, solvents, andsustained-release delivery depots. Formulated glycan-interactingantibodies may be delivered to cells using routes of administrationknown in the art and described herein.

Compositions may also be formulated for direct delivery to organs ortissues in any of several ways in the art including, but not limited to,direct soaking or bathing, via a catheter, by gels, powder, ointments,creams, gels, lotions, and/or drops, by using substrates such as fabricor biodegradable materials coated or impregnated with compositions, andthe like.

Dosing

In some embodiments, the present disclosure provides methods thatinclude administering one or more glycan-interacting antibodies inaccordance with the invention to a subject in need thereof. Nucleicacids encoding glycan-interacting antibodies, proteins or complexes thatinclude glycan-interacting antibodies, or pharmaceutical, imaging,diagnostic, or prophylactic compositions thereof, may be administered toa subject using any amount and any route of administration effective forpreventing, treating, diagnosing, or imaging a disease, disorder, and/orcondition. The exact amount required will vary from subject to subject,depending on the species, age, and general condition of the subject, theseverity of the disease, the particular composition, its mode ofadministration, its mode of activity, and the like. Compositions inaccordance with the invention are typically formulated in dosage unitform for ease of administration and uniformity of dosage. It will beunderstood, however, that the total daily usage of the compositions ofthe present invention will be decided by the attending physician withinthe scope of sound medical judgment. The specific therapeuticallyeffective, prophylactically effective, or appropriate imaging dose levelfor any particular patient will depend upon a variety of factorsincluding the disorder being treated and the severity of the disorder;the activity of the specific compound employed; the specific compositionemployed; the age, body weight, general health, sex and diet of thepatient; the time of administration, route of administration, and rateof excretion of the specific compound employed; the duration of thetreatment; drugs used in combination or coincidental with the specificcompound employed; and like factors well known in the medical arts.

In certain embodiments, compositions in accordance with the presentinvention may be administered at dosage levels sufficient to deliverfrom about 0.0001 mg/kg to about 100 mg/kg, from about 0.01 mg/kg toabout 50 mg/kg, from about 0.1 mg/kg to about 40 mg/kg, from about 0.5mg/kg to about 20 mg/kg, 0.5 mg/kg to about 30 mg/kg, from about 0.01mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, fromabout 2.5 mg/kg to about 5.0 mg/kg, or from about 1 mg/kg to about 25mg/kg, of subject body weight per day, one or more times a day, toobtain the desired therapeutic, diagnostic, prophylactic, or imagingeffect. The desired dosage may be delivered three times a day, two timesa day, once a day, every other day, every third day, every week, everytwo weeks, every three weeks, or every four weeks. In certainembodiments, the desired dosage may be delivered using multipleadministrations (e.g., two, three, four, five, six, seven, eight, nine,ten, eleven, twelve, thirteen, fourteen, or more administrations).

According to the present invention, glycan-interacting antibodies may beadministered in split-dose regimens. As used herein, a “split dose” isthe division of single unit dose or total daily dose into two or moredoses, e.g., two or more administrations of the single unit dose. Asused herein, a “single unit dose” is a dose of any therapeuticadministered in one dose/at one time/single route/single point ofcontact, i.e., single administration event. As used herein, a “totaldaily dose” is an amount given or prescribed in a 24 hr period. It maybe administered as a single unit dose. In one embodiment,glycan-interacting antibodies of the present invention are administeredto a subject in split doses. Glycan-interacting antibodies may beformulated in buffer only or in a formulation described herein.Pharmaceutical compositions including glycan-interacting antibodies asdescribed herein may be formulated into a dosage form described herein,such as a topical, intranasal, intratracheal, or injectable (e.g.,intravenous, intraocular, intravitreal, intramuscular, intracardiac,intraperitoneal or subcutaneous).General considerations in theformulation and/or manufacture of pharmaceutical agents may be found,for example, in Remington: The Science and Practice of Pharmacy 21^(st)ed., Lippincott Williams & Wilkins, 2005 (incorporated herein byreference).

In some embodiments, dosage of glycan-interacting antibodies may beadjusted to reduce bystander effects. As used herein the “bystandereffect” refers to any negative effects on non-target cells or cellsneighboring target cells (also referred to herein as bystander cells).According to such methods, antibody doses or conjugate types may beadjusted to reduce bystander effects. Such adjustments may lead to thetreatments with greater than 95%, greater than 90%, greater than 85%,greater than 80%, greater than 75%, greater than 70%, greater than 65%,greater than 60%, greater than 55%, greater than 50%, greater than 45%,greater than 40%, greater than 35%, greater than 30%, or greater than25% of bystander cells remaining viable.

Coatings or Shells

Solid dosage forms of tablets, dragees, capsules, pills, and granulescan be prepared with coatings and shells such as enteric coatings andother coatings well known in the pharmaceutical formulating art. Theymay optionally include opacifying agents and can be of a compositionthat they release the active ingredient(s) only, or preferentially, in acertain part of the intestinal tract, optionally, in a delayed manner.Examples of embedding compositions which can be used include polymericsubstances and waxes. Solid compositions of a similar type may beemployed as fillers in soft and hard-filled gelatin capsules using suchexcipients as lactose or milk sugar as well as high molecular weightpolyethylene glycols and the like.

IV. Kits and Devices Kits

Any of the compositions described herein may be included in a kit. In anon-limiting example, reagents for generating glycan-interactingantibodies, including antigen molecules are included in a kit. The kitmay further include reagents or instructions for creating orsynthesizing glycan-interacting antibodies. It may also include one ormore buffers. Other kits of the invention may include components formaking glycan-interacting antibody protein or nucleic acid arrays orlibraries and thus, may include, for example, a solid support.

In some embodiments, the present disclosure includes kits for screening,monitoring, and/or diagnosis of a subject that include one or moreglycan-interacting antibodies. Such kits may be used alone or incombination with one or more other methods of screening, monitoring,and/or diagnosis (e.g., as a companion diagnostic). Some kits includeone or more of a buffer, a biological standard, a secondary antibody, adetection reagent, and a composition for sample pre-treatment (e.g., forantigen retrieval, blocking, etc.).

The components of the kits may be packaged either in aqueous media or inlyophilized form. The container means of the kits will generally includeat least one vial, test tube, flask, bottle, syringe or other containermeans, into which a component may be placed, and preferably, suitablyaliquoted. Where there are more than one component in the kit (labelingreagent and label may be packaged together), the kit also will generallycontain a second, third or other additional container into which theadditional components may be separately placed. The kits may alsoinclude a second container means for containing a sterile,pharmaceutically acceptable buffer and/or other diluent. However,various combinations of components may be included in a vial. The kitsof the present invention also will typically include a means forcontaining the glycan-interacting antibodies, e.g., proteins, nucleicacids, and any other reagent containers in close confinement forcommercial sale. Such containers may include injection or blow-moldedplastic containers into which the desired vials are retained.

When the components of the kit are provided in one and/or more liquidsolutions, the liquid solution is an aqueous solution, with a sterileaqueous solution being particularly preferred. However, the componentsof the kit may be provided as dried powder(s). When reagents and/orcomponents are provided as a dry powder, the powder can be reconstitutedby the addition of a suitable solvent. It is envisioned that the solventmay also be provided in another container means. In some embodiments,labeling dyes are provided as a dried powder. It is contemplated that10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 120, 130, 140, 150, 160,170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, 1000 microgramsor at least 1000 micrograms or at most 10 g of dried dye are provided inkits of the invention. The dye may then be resuspended in any suitablesolvent, such as DMSO.

A kit may include instructions for employing the kit components as wellthe use of any other reagent not included in the kit. Instructions mayinclude variations that can be implemented.

Devices

Any of the compositions described herein may be combined with, coatedonto or embedded in a device. Devices include, but are not limited to,dental implants, stents, bone replacements, artificial joints, valves,pacemakers or other implantable therapeutic devices.

V. Equivalents and Scope

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments in accordance with the invention described herein. The scopeof the present invention is not intended to be limited to the aboveDescription, but rather is as set forth in the appended claims.

In the claims, articles such as “a,” “an,” and “the” may mean one ormore than one unless indicated to the contrary or otherwise evident fromthe context. Claims or descriptions that include “or” between one ormore members of a group are considered satisfied if one, more than one,or all of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Theinvention includes embodiments in which more than one, or the entiregroup members are present in, employed in, or otherwise relevant to agiven product or process.

It is also noted that the term “comprising” is intended to be open andpermits but does not require the inclusion of additional elements orsteps. When the term “comprising” is used herein, the term “consistingof” is thus also encompassed and disclosed.

Where ranges are given, endpoints are included. Furthermore, it is to beunderstood that unless otherwise indicated or otherwise evident from thecontext and understanding of one of ordinary skill in the art, valuesthat are expressed as ranges can assume any specific value or subrangewithin the stated ranges in different embodiments of the invention, tothe tenth of the unit of the lower limit of the range, unless thecontext clearly dictates otherwise.

In addition, it is to be understood that any particular embodiment ofthe present invention that falls within the prior art may be explicitlyexcluded from any one or more of the claims. Since such embodiments aredeemed to be known to one of ordinary skill in the art, they may beexcluded even if the exclusion is not set forth explicitly herein. Anyparticular embodiment of the compositions of the invention (e.g., anynucleic acid or protein encoded thereby; any method of production; anymethod of use; etc). can be excluded from any one or more claims, forany reason, whether or not related to the existence of prior art.

All cited sources, for example, references, publications, databases,database entries, and art cited herein, are incorporated into thisapplication by reference, even if not expressly stated in the citation.In case of conflicting statements of a cited source and the instantapplication, the statement in the instant application shall control.

Section and table headings are not intended to be limiting.

EXAMPLES Example 1. Glycan Array Analysis

Optimized glycan arrays are utilized to test antibody affinity andspecificity for multiple glycans in a single experiment. Glycan arraysinclude 71 chemically synthesized and well-defined glycans, most ofwhich are Neu5Ac and Neu5Gc glycan pairs. Array slides are obtainedcommercially (ArrayIt Corp, Sunnyvale, CA) and include the glycanslisted in the following Table.

TABLE 12 Array glycans Glycan ID No. Glycan 1Neu5,9Ac2α2,3Galβ1,4GlcNAcβO(CH2)2CH2NH2 2Neu5Gc9Acα2,3Galβ1,4GlcNAcβO(CH2)2CH2NH2 3Neu5,9Ac2α2,6Galβ1,4GlcNAcβO(CH2)2CH2NH2 4Neu5Gc9Acα2,6Galβ1,4GlcNAcβO(CH2)2CH2NH2 5Neu5Acα2,6GalNAcαO(CH2)2CH2NH2 6 Neu5Gcα2,6GalNAcαO(CH2)2CH2NH2 7Neu5,9Ac2α2,3Galβ1,3GlcNAcβO(CH2)2CH2NH2 8Neu5Gc9Acα2,3Galβ1,3GlcNAcβO(CH2)2CH2NH2 9Neu5,9Ac2α2,3Galβ1,3GalNAcαO(CH2)2CH2NH2 10Neu5Gc9Acα2,3Galβ1,3GalNAcαO(CH2)2CH2NH2 11Neu5Acα2,3Galβ1,4GlcNAcβO(CH2)2CH2NH2 12Neu5Gcα2,3Galβ1,4GlcNAcβO(CH2)2CH2NH2 13Neu5Acα2,3Galβ1,3GlcNAcβO(CH2)2CH2NH2 14Neu5Gcα2,3Galβ1,3GlcNAcβO(CH2)2CH2NH2 15Neu5Acα2,3Galβ1,3GalNAcαO(CH2)2CH2NH2 16Neu5Gcα2,3Galβ1,3GalNAcαO(CH2)2CH2NH2 17Neu5Acα2,6Galβ1,4GlcNAcβO(CH2)2CH2NH2 18Neu5Gcα2,6Galβ1,4GlcNAcβO(CH2)2CH2NH2 19Neu5Acα2,6Galβ1,4GlcβO(CH2)2CH2NH2 20 Νeu5Gcα2,6Galβ1,4GlcβO(CΗ2)2CΗ2ΝΗ221 Neu5Acα2,3Galβ1,4GlcβO(CH2)2CH2NH2 22Neu5Gcα2,3Galβ1,4GlcβO(CH2)2CH2NH2 23 Neu5,9Ac2α2,6GalNAcαO(CH2)2CH2NH224 Neu5Gc9Acα2,6GalNAcαO(CH2)2CH2NH2 25 Neu5Acα2,3GalβO(CH2)2CH2NH2 26Νeu5Gcα2,3GalβO(CΗ2)2CΗ2ΝΗ2 27 Neu5Acα2,6GalβO(CH2)2CH2NH2 28Neu5Acα2,6GalβO(CH2)2CH2NH2 29 Neu5,9Ac2α2,3GalβO(CH2)2CH2NH2 30Neu5Gc9Acα2,3GalβO(CH2)2CH2NH2 31 Neu5,9Ac2α2,6GalβO(CH2)2CH2NH2 32Neu5Gc9Acα2,6GalβO(CH2)2CH2NH2 33 Neu5Acα2,3Galβ1,3GalNAcβO(CH2)2CH2NH234 Neu5Acα2,3Galβ1,3GalNAcβO(CH2)2CH2NH2 35Neu5,9Ac2α2,3Galβ1,3GalNAcβO(CH2)2CH2NH2 36Neu5Gc9Acα2,3Galβ1,3GalNAcβO(CH2)2CH2NH2 37Neu5,9Ac2α2,6Galβ1,4GlcβO(CH2)2CH2NH2 38Neu5Gc9Acα2,6Galβ1,4GlcβO(CH2)2CH2NH2 39Neu5,9Ac2α2,3Galβ1,4GlcβO(CH2)2CH2NH2 40Neu5Gc9Acα2,3Galβ1,4GlcβO(CH2)2CH2NH2 41Neu5Acα2,8Neu5Acα2,3Galβ1,4GlcβO(CH2)2CH2NH2 42Neu5Acα2,8Neu5Acα2,8Neu5Acα2,3Galβ1,4GlcβO(CH2)2CH2 NH2 43Galβ1,4GlcβO(CH2)2CH2NH2 45 Galβ1,4GlcNAcβO(CH2)2CH2NH2 47GalNAcαO(CH2)2CH2NH2 51 Galβ1,3GalNAcβO(CH2)2CH2NH2 52Galβ1,3GlcNAcαO(CH2)2CH2NH2 53 Galβ1,3GlcNAcβO(CH2)2CH2NH2 54Galβ1,4GlcNAc6SβO(CH2)2CH2NH2 55Neu5Acα2,3Galβ1,4(Fucα1,3)GlcNAcβO(CH2)2CH2NH2 56Neu5Gcα2,3Galβ1,4(Fucα1,3)GlcNAcβO(CH2)2CH2NH2 57Neu5Acα2,3Galβ1,4(Fucα1,3)GlcNAc6SβO(CH2)2CH2NH2 58Neu5Gcα2,3Galβ1,4(Fucα1,3)GlcNAc6SβO(CH2)2CH2NH2 59Galβ1,3GlcNAcβ1,3Galβ1,4GlcβO(CH2)2CH2NH2 60Neu5Acα2,3Galβ1,3GlcNAcβ1,3Galβ1,4GlcβO(CH2)2CH2NH2 61Neu5Gcα2,3Galβ1,3GlcNAcβ1,3Galβ1,4GlcβO(CH2)2CH2NH2 62Neu5Acα2,3Galβl,4GlcNAc6SβO(CH2)2CH2NH2 63Neu5Gcα2,3Galβ1,4GlcNAc6SβO(CH2)2CH2NH2 64Neu5Acα2,8Neu5Acα2,3Galβ1,4GlcβO(CH2)3NHCOCH2(OCH 2CH2)6NH2 65Neu5Acα2,8Neu5Acα2,8Neu5Acα2,3Galβ1,4GlcβO(CH2)3NHC OCH2(OCH2CH2)6NH2 66Neu5Acα2,6(Neu5Acα2,3)Galβ1,4GlcβO(CH2)2CH2NH2 67Neu5Acα2,6(Neu5Gcα2,3)Galβ1,4GlcβO(CH2)2CH2NH2 68Neu5Acα2,6(KDNα2,3)Galβ1,4GlcβO(CH2)2CH2NH2 69Neu5Gcα2,8Neu5Acα2,3Galβ1,4GlcβO(CH2)2CH2NH2 70KDNα2,8Neu5Acα2,3Galβ1,4GlcβO(CH2)2CH2NH2 71Neu5Acα2,8Kdnα2,6Galβ1,4GlcβO(CH2)2CH2NH2 72Neu5Acα2,8Neu5Gcα2,3Galβ1,4GlcβO(CH2)2CH2NH2 73Neu5Acα2,8Neu5Gcα2,6Galβ1,4GlcβO(CH2)2CH2NH2 74KDNα2,8Neu5Gcα2,3Galβ1,4GlcβO(CH2)2CH2NH2 75Neu5Gcα2,8Neu5Gcα2,3Galβ1,4GlcβO(CH2)2CH2NH2 76Neu5Acα2,8Neu5Acα2,6Galβ1,4GlcβO(CH2)2CH2NH2

300 ml of epoxy blocking buffer is prepared by combining 15 ml of 2 MTris buffer (pH 8) with 0.9 ml of 16.6 M ethanolamine and 284.1 ml ofdistilled water. The solution is brought to a final pH of 9.0 with HCl.The solution is filtered using a 0.2 µM nitrocellulose membrane. Theepoxy buffer solution as well as 1 L of distilled water are prewarmed to50° C. Glass slides are arranged in a slide holder and quickly submergedin a staining tub with the warmed epoxy blocking buffer. Slides areincubated in the epoxy blocking buffer for 1 hour at 50° C. withperiodic shaking to deactivate epoxy binding sites. Next, slides arerinsed and blocked with PBS with 1% OVA at 25° C. for one hour. Serumsamples with polyclonal antibodies (1:1000) or purified monoclonalantibodies (1 ug/mL), are diluted in PBS with 1% OVA and added to theglycan array for one hour at 25° C. After extensive washing, binding ofantibodies are detected by incubating glycan microarray slides withCy3-conjugated anti-mouse IgG (Jackson Immunoresearch, West Grove, PA)for one hour. Slides are then washed extensively, dried and scanned witha Genepix 4000B scanner (Laser at 100%; gain at 350; 10 µm pixels). Rawdata from scanned images are extracted using the Genepix software andanalysis of raw data is carried out. Antibodies are considered to behighly specific for AcSTn and GcSTn if they demonstrate binding to bothmolecules, but not to Tn or any other glycans on the array.

Based on array analysis, antibodies are classified according to arrayglycan binding profile. Antibodies are classified as “Group 1”antibodies, capable of binding AcSTn and GcSTn, if they bind to glycans5, 6, 23 and 24. Such antibodies are referred to as Pan-STn antibodiesdue to their ability to associate with a wider range of STn structuresand the portion of STn indicated by the large oval in FIG. 1A.Antibodies are classified as “Group 2” antibodies, capable of bindingSTn as well as some related structures that include an O-linkage toserine or threonine, if they bind to glycans 5, 6, 23, 24, 27 and 31.These antibodies are thought to associate with the portion of STnindicated by the large oval in FIG. 1B. Some Group 2 antibodiespreferably bind to structures with AcSTn over structures with GcSTn.Antibodies are classified as “Group 3” antibodies (capable of bindingSTn, but may also bind a broader set of related structures) if they bindglycans 5, 6, 23, 24, 17, 3, 19, 37, 27 and 31. Unlike Group 2antibodies, Group 3 antibodies do not require that such structures havean O-linkage to serine or threonine. Group 3 antibodies are thought toassociate with the portion of STn indicated by the large oval in FIG.1C. Finally, antibodies are “Group 4” antibodies, capable of binding toboth AcSTn and GcSTn as well as the un-sialylated Tn antigen (thereforehaving broader specificity) if they bind to glycans 5, 6, 23, 24 and 47.Group 4 antibodies are thought to associate with the portion of STnindicated by the large oval in FIG. 1D.

Example 2. Flow Cytometry-Based Analysis of Antibody Binding

Flow cytometry-based analysis is carried out to elucidate thedose-response curve for binding of antibodies to cell surface antigens.For these analyses, various cell lines are employed.

MDA-MB-231 cells are human breast cancer cells. They are grown inEarle’s Minimum Essential Medium supplemented with 10% fetal calf serum(FCS), 100 µg/ml penicillin, 100 UI/ml streptomycin and 45 µg/mlgentamycin. MCF-7 cells are also human breast cancer cells and are grownunder the same conditions as MDA-MB-231 cells. Stably transfectedversions of MDA-MB-231 (MDA-MB-231-STn, clone TAH3.P10) and MCF-7 cells(clone A12.1 for MCF-7 cells) that over express(Alpha-N-Acetyl-Neuraminyl-2,3-Beta-Galactosyl-1,3)-N-AcetylgalactosaminideAlpha-2,6-Sialyltransferase I (GalNAc α2,6-sialyltransferase I orST6GalNAc I), are also cultured under the same conditions with theexception of an added 1 mg/ml of G418 to support cells expressing thetransgene. ST6GalNAc I is an enzme capable of sialylating GalNAc. As aresult of over expression, transfected cells express high levels ofNeu5Ac-STn (see Julien, S. et al., Glycoconjugate journal. 2001. 18,883-93; the contents of which are herein incorporated by reference intheir entirety).

E3 cells are murine breast cancer cells. They are cultured in Dulbecco’sE4 medium with 10% FCS. Stably transfected versions of E3 cellsexpressing high levels of Neu5Gc-STn (E3-STn) are cultured with 600µg/ml of G418 and 200 µg/ml hygromycin. During growth and maintenance ofexperimental cells, trypsin is not used for cell passaging.

OV90 and OVCAR3 cells are also used. These are human ovarian cancer celllines, described previously.

SNU-16 cells are also used. These are gastric cancer cell lines thatexpress low levels of STn.

For analysis, cells are harvested using StemPro Accutase (LifeTechnologies, Carlsbad, CA) and washed with PBS including 5% FBS beforepelleting by light centrifugation. Cell numbers and viability aredetermined by trypan blue dye exclusion analysis and cell concentrationsare adjusted to 5 × 10⁶ cells/ml in PBS with 5% FBS. 50 µl of cells areadded to each well of an assay plate. Cells are combined with 50 µlsolutions of antibody being analyzed or control antibodies and incubatedfor 1 hour at 4° C. Cells are washed and pelleted twice with PBS with 5%FBS before being treated with 100 µl of PBS with 5% FBS including a1:1,500 dilution of anti-mouse IgG (Southern Biotech, Birmingham,Alabama,) conjugated to allophycocyanin (APC). Cells are incubated for30 min at 4° C. before washing and resuspending in 200 µl of propidiumiodide (PI) diluted 1:1000 in PBS with 5% FBS . Treated cells are thensubjected to flow cytometry analysis and 10,000 events are acquired foreach sample.

Example 3. Antibody Humanization

Fully humanized heavy and light chains are designed with CDRs presentedherein. Protein models of the variable regions are generated usingexisting antibody structures as templates. Segments of starting heavyand light chain variable region amino acid sequences are compared withhuman sequences for possible inclusion in the fully humanized sequences.Series of humanized heavy and light chain variable regions are designedentirely from segments of human variable region sequences with theobjective that T cell epitopes be avoided. Variant human sequencesegments with significant incidence of potential T cell epitopes asdetermined by in silico technologies are discarded.

Humanized heavy and light chain variable region genes are constructedfrom overlapping oligonucleotides assembled into full length genes usingthe ligase chain reaction (LCR). LCR products are amplified and suitablerestriction sites are added for cloning into expression vectors. PCRproducts are cloned into intermediate vectors and confirmed bysequencing.

For construction of expression plasmids encoding fully humanizedantibodies with human constant regions, DNA sequences for each variableregion are inserted into mammalian expression vectors between anupstream cytomegalovirus immediate/early promoter/enhancer (CMV IE) plusthe immunoglobulin signal sequence and a downstream immunoglobulinconstant region gene. DNA samples are prepared for transfection intomammalian cells.

For generation of cell lines and selection of lead fully humanizedantibodies, heavy and light chain plasmid DNA pairs are transfected intomammalian cells (NS0). Cell lines producing humanized antibodies areexpanded and antibody samples are purified. Antibodies are tested inprimary and secondary binding assays to determine leading antibodycandidates. The 3 leading candidates are used for further analysis.

Example 4. Immunogenicity Testing

Lead antibodies are subjected to EpiScreen (Antitope, Paradise Valley,AZ) whole antibody human T cell assays using a minimum of 20 bloodsamples from healthy volunteer donors. Immunogenicity of lead antibodiesis compared with control chimeric antibodies with starting antibodyvariable regions and matched human constant regions. Data arebenchmarked against EpiScreen whole protein data for clinical-stagebiologics.

Example 5. Antibody Sequence Analysis

Anti-glycan antibody variable domain sequences were analyzed forsequence similarities as well as for characteristics that may impactantibody function, expression, stability or immunogenicity. Theantibodies used were commercially available or developed previously asdescribed in U.S. Publication Nos. US2016/0264684 and US2016/0130356,the contents of which are herein incorporated by reference in theirentirety. Analysis revealed far more variability in the light chainvariable domains as compared to the heavy chain variable domains.Additionally, it was determined that heavy chain variable domains of theanti-glycan antibodies originated from one germline gene, muIGHV1S53, agermline gene that is shared with anti-STn antibodies known in the art:antibody 3F1 (SBH Sciences, Natick, MA), antibody B72.3 (see Colcher, D.et al., 1981. PNAS. 78(5): 3199-203), and antibody CC49 (see Muraro, R.et al., 1988. Cancer Res. 48: 4588-96). A comparative view of heavychain CDR sequences based on the analysis is presented in the followingTable.

TABLE 13 CDR sequence heavy chain comparison Clone ID CDR-H1 SEQ ID NOCDR-H2 SEQ ID NO CDR-H3 SEQ ID NO 8C2-2D6 GYTFTDHAIH 105YISPGNGDIKYNEKFKG 107 SITTSY 114 4G8-1E3 GYIFTDHAIH 106YISPGNGDIKYNEKFKG 107 SITTSY 114 2G12-2B2 GYTFTDHAIH 105YFSPGNDDIKYNEKFRG 108 SLSTPY 115 5G2-1B3 GYTFTDHAIH 105YFSPGNDDIKYNEKFKV 109 SYYGD 116 5E6-2E7 GYTFTDHAIH 105 YISPGNGDIKYNEKFKV110 SITTPY 117 2C2-2C5 GYTFTDHAIH 105 YISPGNGDIKYNEKFKG 107 SITTPY 1179F11-1F7 GYTFTDHAIH 105 YISPGNGDIKYNEKFKV 110 SITTPY 117 1F6-1C10GYTFTDHAIH 105 YISPGNGDVKYSERFKG 137 SLSTPY 115 7D3-2C10 GYTFTDHAIH 105YFSPGNDDIKYSEKFKG 138 SITTPY 117 7A5-2G12 GYTFTDHAIH 105YISPGNDDIKYNEKFKG 113 SITTSY 114 10F4-2A9 GYTFTDHAIH 105YISPGNGDIKYDEKFKG 139 SITTSY 114 2F4-1E2 GYTFTDHAIH 105YISPGNGDIKYNEKFKG 107 QLGQGY 140 2C6-2F11 GYTFSDHAIH 136YISPGNDDIKYNEKFKG 113 SMIGVY 141 6B11-2E3 GYTFTDHAIH 105YISPGNDDIKYNEKFKG 113 SITTSY 114 3F1 GYTFTDHAIH 105 YISPGNGDIKYNEKFKD111 SLLALD Y 118 CC49 GYTFTDHAIH 105 YFSPGNDDFKYNEKFKG 112 SLNMAY 119B72.3 GYTFTDHAIH 105 YISPGNDDIKYNEKFKG 113 SYYGH 120 ConsensusGYTFTDHAIH 105 YISPGNGDIKYNEKFKG 107 SITTSY 114

CDR-H3 sequences varied by plus or minus one amino acid relative to themedian length.

Interestingly, target-specific light chains were found to be derivedfrom 5 light chain germline families: IGKV6, IGKV15, IGKV8, IGKV1 andIGKV12. Of these, all had the same CDR-L2 and CDR-L3 sequence lengths.Two classes of CDR-L1 sequences were found to persist [long (IGKV8 andIGKV1) and short (IGKV6, IGKV15, and IGKV12)], potentially presentingunified topology in each class.

A comparison of light chain CDR sequences is presented in the followingTable.

TABLE 14 CDR sequence light chain comparison Clone ID CDR-L1 SEQ ID NOCDR-L2 SEQ ID NO CDR-L3 SEQ ID NO 8C2-2D6 KASENVVTYVS 121 GASNRYT 77GQGYSYPYT 89 8C2-2D6(V2) HASQNINVWLS 142 KASNLYT 147 QHDQSYPTY 1484G8-1E3 HASQHINFWLS 122 KASNLHT 80 QQDQSYPYM 103 2G12-2B2KSSQSLLNRGNHKNYLT 123 WASTRES 85 QNDYTYPYT 97 5G2-1B3 RASENIYSHLA 124GATNLAD 79 QHFWGAPFT 91 5E6-2E7 KSSQSLLNSGKTKNYLT 125 WASTRES 85KNDYSYPYT 102 2C2-2C5 KASQSVNNNVA 126 YASNRYT 84 QQGYSSPWT 96 1F6-1C10KSSQSLLNSGNQKSYLT 143 WASTRDS 83 QSDYSYPYT 95 7D3-2C10 HASQNINVWLS 142KVSNLHT 88 QQDQSYPYT 101 7A5-2G12 KASENVVIYVS 144 GASNRYT 77 GQGYSYPYT89 10F4-2A9 KASENVVTYVS 121 GASNRYT 77 GQGYSYPYT 89 2F4-1E2RSSQSLVHSYGNTYLH 145 KVSNRFS 81 SQNTHVPYT 93 2C6-2F11 RFSQSLVQSNGNTYLQ146 KVSNRFC 86 SQSTHAPLT 98 6B11-2E3 KASENVVTYVS 121 GASNRYT 77GQGYSYPYT 89 3F1 KASQDVGTNIA 127 SASTRHT 130 QQYSSFPLT 133 CC49KSSQSLLYSGNQKNYLA 128 WASARES 131 QQYYSYPLT 134 B72.3 RASENIYSNLA 129AATNLAD 132 QHFWGTPYT 135

Taken together, the sequence analysis suggests distinct patterns ofCDR-H3 diversity that correspond with specific light chain germlinepairings. Three sequence groups [Group A (with subgroups A1 and A2),Group B (with subgroups B1 and B2), and Group C] were identified basedon these pairings. A listing of antibodies falling into each group arepresented in the following Table.

TABLE 15 Antibody sequence groups Clone ID Light Chain Murine GermlineSequence Group 8C2-2D6 IGKV6-20 Group A1 7A5-2G12 IGKV6-20 Group A110F4-2A9 IGKV6-20 Group A1 6B11-2E3 IGKV6-20 Group A1 2C2-2C5 IGKV6-32Group A1 3F1 IGKV6-32 Group A1 4G8-1E3 IGKV15-103 Group A2 7D3-2C10IGKV15-103 Group A2 8C2-2D6(V2) IGKV15-103 Group A2 2G12-2B2 IGKV8-19Group B1 5E6-2E7 IGKV8-19 Group B1 1F6-1C10 IGKV8-19 Group B1 CC49IGKV8-30 Group B1 2F4-1E2 IGKV1-110 Group B2 2C6-2F11 IGKV1-110 Group B25G2-1B3 IGKV12-46 Group C B72.3 IGKV12-46 Group C

Group A includes antibodies 8C2-2D6, 4G8-1E3 and 3F1. These antibodieshave similar CDR-H3 sequences, with the exception of 3F1, which isdistinct from all other antibodies in terms of CDR-H3 length (having anextra amino acid, creating a longer loop). Group A antibodies also havelight chain CDRs with similarities, especially in CDR residue lengths.

Group B includes antibodies 2G12-2B2 and CC49. Among the similarities inheavy chain sequences, these antibodies have conserved F and D residuesin the CDR-H2 and a conserved L residue in the CDR-H3. Additionally,Group B antibodies have highly similar light chain sequences.

Group C antibodies include 5G2-1B3 and B72.3. Among the similaritiesbetween their heavy chain sequences, these antibodies have conserved Dresidues in their CDR-H2 sequences as well as a YYG motif in theirCDR-H3 sequences. Group C antibodies also have highly similar lightchain sequences.

The limited number of groups identified highlights the relatively raresequence specificity necessary for anti-STn binding. Antibody groupingfacilitates the identification of relevant intra-group sequence-basedcontributions to epitope binding. Notably, within Group A, 3F1 uniquelycontains an extended CDR-H3 loop that may contribute to a novel bindingprofile. Interestingly, immunohistochemistry data indicates that 3F1 maybind to a broader range of targets, including undesired binding toendothelial cells.

Example 6. Antibody Variants

Variable domain sequences for anti-glycan antibodies of the inventionwere analyzed for sequence characteristics that may impact antibodyfunction, expression, stability and/or immunogenicity.

Many of the antibodies analyzed had CDR-H2 sequences containing NGresidue pairs, making them susceptible to asparagine deamidation, withpossible conversion to glutamate and pyroglutamate in a 3:1 ratio overtime. These sequences may be subjected to mutagenesis to convert NGresidue pairs to SG or QG pairs to prevent deamidation at these sites.Alternatively, these antibodies may be formulated to reduce deamidation.

Antibodies 2B2-2A7 and 5G2-1B3 had aspartate isomerization sites(identified by DG amino acid residue pairs) in their light chainvariable domains. Aspartic acid at these sites can convert intoglutamate and pyroglutamate in a 3:1 ratio over time. These sequencesmay be subjected to mutagenesis to convert DG residue pairs to SG or QGto prevent isomerization at these sites. Alternatively, these antibodiesmay be formulated to reduce isomerization.

Many of the antibodies have heavy chains with N-terminal glutamineresidues. These sequences may be subjected to mutagenesis to convertN-terminal glutamine residues to glutamate residues.

Sequence analysis for aggregation-prone patches revealed an HFW segmentin the CDR-L3 of 5G2-1B3, which carries some risk of increasing antibodyaggregation. Aggregation stability studies may be carried out withvariants of this motif to identify less aggregation-prone antibodies.

Example 7. Antibody Humanization

Humanized versions of lead antibodies were developed using sequence andstructural analysis. First, mouse germline antibody sequences wereidentified for each antibody (see the following Table).

TABLE 16 Antibody mouse germline sequences Antibody VH mouse germline VLmouse germline 4G8-1E3 muIGHV1S53 muIGKV15-103 5G2-1B3 muIGHV1S53muIGKV12-46 2G12-2B2 muIGHV1S53 muIGKV8-19 8C2-2D6 muIGHV1S53 muIGKV6-203F1 muIGHV1S53 muIGKV6-23

Antibody variable domain sequences were then compared to human frameworksequences and human framework sequences suitable for CDR grafting wereidentified by homology. A schematic of a variable domain is shown inFIG. 2 , demonstrating the layout of antibody variable domain frameworkregions [framework region 1 (FR1), framework region 2 (FR2), frameworkregion 3 (FR3) and framework region 4 (FR4)] in relation to CDRs. Thefollowing Table indicates the human framework or human consensussequence selected to replace the corresponding framework region ofantibodies 4G8-1E3, 5G2-1B3, 2G12-2B2, 8C2-2D6, and 3F1. FR4 of humanconsensus 1 heavy chain corresponds to the amino acid sequenceWGQGTLVTVSS (SEQ ID NO: 215) and FR4 of human consensus 1 light chaincorresponds to the amino acid sequence FGQGTKVEIK (SEQ ID NO: 216).

TABLE 17 Selected human framework regions mAb Chain FR1 CDR1 (SEQ ID NO)FR2 CDR2 (SEQ ID NO) FR3 CDR3 (SEQ ID NO) FR4 4G8-1E3 VH IGHV1-18^(∗)01106 IGHV1-18^(∗)01 107 IGHV1-18^(∗)01 114 Human Consensus 1, Heavy Chain4G8-1E3 VL IGKV1-39^(∗)01 122 IGKV1-39^(∗)01 80 IGKV1-39^(∗)*01 103Human Consensus 1, Light Chain 5G2-1B3 VH IGHV1-18^(∗)01 105IGHV1-18^(∗)01 109 IGHV1-18^(∗)01 116 Human Consensus 1, Heavy Chain5G2-1B3 VL IGKV1-39^(∗)01 124 IGKV1-39^(∗)01 79 IGKV1-39^(∗)01 91 HumanConsensus 1, Light Chain 2G12-2B2 VH IGHV1-18^(∗)01 105 IGHV1-18^(∗)01108 IGHV1-18^(∗)01 115 Human Consensus 1, Heavy Chain 2G12-2B2 VLIGKV4-1^(∗)01 123 IGKV4-1^(∗)01 85 IGKV4-1^(∗)01 97 Human Consensus 1,Light Chain 8C2-2D6 VH IGHV1-18^(∗)01 105 IGHV1-18^(∗)01 107IGHV1-18^(∗)01 114 Human Consensus 1, Heavy Chain 8C2-2D6 VLIGKV1-39^(∗)01 121 IGKV1-39^(∗)01 77 IGKV1-39^(∗)01 89 Human Consensus1, Light Chain 8C2-2D6 VL (V2) IGKV1-39^(∗)01 142 IGKV1-39^(∗)01 147IGKV1-39^(∗)*01 148 Human Consensus 1, Light Chain 3F1 VH IGHV1-18^(∗)01105 IGHV1-18^(∗)01 111 IGHV1-18^(∗)01 118 Human Consensus 1, Heavy Chain3F1 VL IGKV1-39^(∗)01 127 IGKV1-39^(∗)01 130 IGKV1-39^(∗)01 133 HumanConsensus 1, Light Chain

Additional analysis was conducted to identify residues that may beback-crossed to improve antibody binding or other properties. Based onthis analysis, several humanized VL and VH sequences were designed forsynthesis and testing. These include the variable domain sequencespresented in the following Table. In the Table, VH or VL domains areindicated, followed by a digit to show the variant number. Domains withthe digit “0” represent the humanized sequence without anyback-mutation.

TABLE 18 Humanized variable domains mAb Chain Sequence SEQ ID NO 5G2-1B3VL0 DIQMTQSPSSLSASVGDRVTITCRASENIYSHLAWYQQKPGKAPKLLIYGATNLADGVPSRFSGSGSGTDFTLT ISSLQPEDFATYYCQHFWGAPFTFGQGTKVEIK217 5G2-1B3 VL1 DIQMTQSPSSLSASVGDRVTITCRASENIYSHLAWYQQKPGKAPKLLVYGATNLASGVPSRFSGSGSGTQFTL TISSLQPEDFATYYCQHFWGAPFTFGQGTKVEIK218 5G2-1B3 VL2 DIQMTQSPSSLSASVGDRVTITCRASENIYSHLAWYQQKPGKAPKLLVYGATNLADGVPSRFSGSGSGTQFTL TISSLQPEDFATYYCQHFWGAPFTFGQGTKVEIK219 5G2-1B3 VH0 QVQLVQSGAEVKKPGASVKVSCKASGYTFTDHAIHWVRQAPGQGLEWMGYFSPGNDDIKYNEKFKVRVT MTTDTSTSTAYMELRSLRSDDTAVYYCARSYYGDWGQGTLVTVSS 220 5G2-1B3 VH1 QVQLVQSGAEVKKPGASVKVSCKASGYTFTDHAIHWVRQAPGQGLEWMGYFSPGNDDIKYNEKFKVRVT MTADKSSSTAYMELRSLRSDDTAVYFCKRSYYGDWGQGTLVTVSS 221 5G2-1B3 VH2 QVQLVQSGAEVKKPGASVKISCKASGYTFTDHAIHWVRQAPGQGLEWIGYFSPGNDDIKYNEKFKVRATLTA DKSSSTAYMELRSLRSDDTAVYFCKRSYYGDWGQGTLVTVSS 222 5G2-1B3 VH3 EVQLVQSGAEVKKPGASVKVSCKASGYTFTDHAIHWVRQAPGQGLEWMGYFSPGNDDIKYNEKFKVRVT MTADKSSSTAYMELRSLRSDDTAVYFCKRSYYGDWGQGTLVTVSS 223 5G2-1B3 VH4 EVQLVQSGAEVKKPGASVKISCKASGYTFTDHAIHWVRQAPGQGLEWIGYFSPGNDDIKYNEKFKVRATLTA DKSSSTAYMELRSLRSDDTAVYFCKRSYYGDWGQGTLVTVSS 224 4G8-1E3 VL0 DIQMTQSPSSLSASVGDRVTITCHASQHINFWLSWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTL TISSLQPEDFATYYCQQDQSYPYMFGQGTKVEIK225 4G8-1E3 VL1 DIQMTQSPSSLSASVGDRVTITCHASQHINFWLSWYQQKPGKIPKLLIYKASNLHTGVPSRFSGSGSGTGFTL TISSLQPEDFATYYCQQDQSYPYMFGQGTKVEIK226 4G8-1E3 VL2 DIQMTQSPSSLSASVGDRITITCHASQHINFWLSWYQQKPGKIPKLLIYKASNLHTGVPSRFSGSGSGTGFTLTI SSLQPEDVATYYCQQDQSYPYMFGQGTKLEIK227 4G8-1E3 VL3 DIQMTQSPSSLSASVGDRVTITCHASQHINFWLSWYQQKPGKIPKLLIYKASNLHTGVPSRFSGSGSGTGFTL TISSLQPEDFATYYCQQDQSYPYFFGQGTKVEIK228 4G8-1E3 VL4 DIQMTQSPSSLSASVGDRITITCHASQHINFWLSWYQQKPGKIPKLLIYKASNLHTGVPSRFSGSGSGTGFTLTI SSLQPEDVATYYCQQDQSYPYFFGQGTKLEIK229 4G8-1E3 VH0 QVQLVQSGAEVKKPGASVKVSCKASGYIFTDHAIHWVRQAPGQGLEWMGYISPGNGDIKYNEKFKGRVT MTTDTSTSTAYMELRSLRSDDTAVYYCARSITTSYWGQGTLVTVSS 230 4G8-1E3 VH1 QVQLVQSGAEVKKPGASVKVSCKASGYIFTDHAIHWVRQAPGQGLEWMGYISPGNGDIKYNEKFKGRVT MTADKSSSTAYMELRSLRSDDTAVYFCKRSITTSYWGQGTLVTVSS 231 4G8-1E3 VH2 QVQLVQSGAEVKKPGASVKISCKASGYIFTDHAIHWVRQAPGQGLEWIGYISPGNGDIKYNEKFKGRATLTADKSSSTAYMHLRSLRSDDTAVYFCKRSITTSYWGQG TLVTVSS 232 4G8-1E3 VH3EVQLVQSGAEVKKPGASVKVSCKASGYIFTDHAIHW VRQAPGQGLEWMGYISPGSGDIKYNEKFKGRVTMTADKSSSTAYMELRSLRSDDTAVYFCKRSITTSYWGQ GTLVTVSS 233 4G8-1E3 VH4EVQLVQSGAEVKKPGASVKISCKASGYIFTDHAIHWVRQAPGQGLEWIGYISPGSGDIKYNEKFKGRATLTADKSSSTAYMHLRSLRSDDTAVYFCKRSITTSYWGQG TLVTVSS 234 2G12-2B2 VL0DIVMTQSPDSLAVSLGERATINCKSSQSLLNRGNHKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYTYPYTFGQGT KVEIK 235 2G12-2B2 VL2DIVMTQSPDSLAVSLGERVTMSCKSSQSLLNRGNHKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYTYPYTFGQGT KVEIK 236 2G12-2B2 VH0QVQLVQSGAEVKKPGASVKVSCKASGYTFTDHAIH WVRQAPGQGLEWMGYFSPGNDDIKYNEKFRGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARSLSTPYW GQGTLVTVSS 237 2G12-2B2 VH1QVQLVQSGAEVKKPGASVKVSCKASGYTFTDHAIH WVRQAPGQGLEWMGYFSPGNDDIKYNEKFRGRVTMTADKSSSTAYMELRSLRSDDTAVYFCKRSLSTPYW GQGTLVTVSS 238 2G12-2B2 VH2QVQLVQSGAEVKKPGASVKISCKASGYTFTDHAIHWVRQAPGQGLEWIGYFSPGNDDIKYNEKFRGRVTLTADKSSSTAYMELRSLRSDDTAVYFCKRSLSTPYWGQG TLVTVSS 239 2G12-2B2 VH3EVQLVQSGAEVKKPGASVKVSCKASGYTFTDHAIH WVRQAPGQGLEWMGYFSPGNDDIKYNEKFRGRVTMTADKSSSTAYMELRSLRSDDTAVYFCKRSLSTPYW GQGTLVTVSS 240 2G12-2B2 VH4EVQLVQSGAEVKKPGASVKISCKASGYTFTDHAIHWVRQAPGQGLEWIGYFSPGNDDIKYNEKFRGRVTLTADKSSSTAYMELRSLRSDDTAVYFCKRSLSTPYWGQG TLVTVSS 241 8C2-2D6 VL0DIQMTQSPSSLSASVGDRVTITCKASENVVTYVSWYQQKPGKAPKLLIYGASNRYTGVPSRFSGSGSGTDFTL TISSLQPEDFATYYCGQGYSYPYTFGQGTKVEIK242 8C2-2D6 VL1 NIQMTQSPSSLSASVGDRVTITCKASENVVTYVSWYQQKPGKAPKLLIYGASNRYTGVPSRFSGSGSATDFTL TISSLQPEDFATYYCGQGYSYPYTFGQGTKVEIK243 8C2-2D6 VL2 NIVMTQSPSSMSMSVGDRVTLTCKASENVVTYVSWYQQKPGKSPKLLIYGASNRYTGVPSRFSGSGSATDFTLTISSVQPEDLATYHCGQGYSYPYTFGQGTKLEIK 244 8C2-2D6(V2) VL0DIQMTQSPSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKAPKLLIYKASNLYTGVPSRFSGSGSGTDFTL TISSLQPEDFATYYCQHDQSYPYTFGQGTKVEIK245 8C2-2D6(V2) VL1 DIQMTQSPSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKIPKLLIYKASNLYTGVPSRFSGSGSGTGFTL TISSLQPEDFATYYCQHDQSYPYTFGQGTKVEIK246 8C2-2D6(V2) VL2 DIQMTQSPSSLSASVGDRITITCHASQNINVWLSWYQQKPGKIPKLLIYKASNLYTGVPSRFSGSGSGTGFTLTI SSLQPEDFATYYCQHDQSYPYTFGQGTKLEIK247 8C2-2D6(V2) VL3 DIQMNQSPSSLSASVGDRITITCHASQNINVWLSWYQQKPGKIPKLLIYKASNLYTGVPSRFSGSGSGTGFTLTI SSLQPEDFATYYCQHDQSYPYTFGQGTKLEIK248 8C2-2D6 VH0 QVQLVQSGAEVKKPGASVKVSCKASGYTFTDHAIHWVRQAPGQGLEWMGYISPGNGDIKYNEKFKGRVT MTTDTSTSTAYMELRSLRSDDTAVYYCARSITTSYWGQGTLVTVSS 249 8C2-2D6 VH1 QVQLVQSGAEVKKPGASVKVSCKASGYTFTDHAIHWVRQAPGQGLEWMGYISPGNGDIKYNEKFKGRVT MTADKSSTTAYMELRSLRSDDTAVYFCKRSITTSYWGQGTLVTVSS 250 8C2-2D6 VH2 QVQLVQSGAEVKKPGASVKISCKASGYTFTDHAIHWVRQAPGQGLEWIGYISPGNGDIKYNEKFKGRATLTADKSSTTAYMELRSLRSDDTAMYFCKRSITTSYWGQG TLVTVSS 251 8C2-2D6 VH3EVQLVQSGAEVKKPGASVKVSCKASGYTFTDHAIH WVRQAPGQGLEWMGYISPGSGDIKYNEKFKGRVTMTADKSSTTAYMELRSLRSDDTAVYFCKRSITTSYWG QGTLVTVSS 252 8C2-2D6 VH4EVQLVQSGAEVKKPGASVKISCKASGYTFTDHAIHWVRQAPGQGLEWIGYISPGSGDIKYNEKFKGRATLTADKSSTTAYMELRSLRSDDTAMYFCKRSITTSYWGQG TLVTVSS 253

Variable domain pairs were selected for initial expression of fullantibodies and testing. Among the pairs selected for 5G2-1B3 were VL0and VH0 (no back-mutation); VL1 and VH1; VL1 and VH2; VL2 and VH1; VL2and VH2; and VL1 and VH3. Among the pairs selected for 4G8-1E3 were VL0and VH0 (no back-mutation); VL1 and VH1; VL1 and VH2; VL2 and VH1; VL2and VH2; VL1 and VH3; VL3 and VH1; and VL3 and VH3. Among the pairsselected for 2G12-2B2 were VL0 and VH0 (no back-mutation); VL0 and VH1;VL0 and VH2; VL2 and VH1; VL2 and VH2; and VL0 and VH3. Among the pairsselected for 8C2-2D6 were VL0 and VH0 (no back-mutation); VL1 and VH1;VL1 and VH2; VL2 and VH1; VL2 and VH2; and VL1 and VH3. Among the pairsselected for 8C2-2D6(V2) were VL0 and VH0 (no back-mutation); VL1 andVH1; VL1 and VH2; VL2 and VH1; VL2 and VH2; VL3 and VH2; and VL1 andVH3.

3F1 full length heavy chain amino acid sequence (SEQ ID NO: 40) wasassessed for the presence of unpaired cysteine residues. Residue 80 ofthe heavy chain was identified as a cysteine that would be unpaired whenpart of an IgG. The cysteine was determined to be accessible to solventwhen in solution and therefore reactive. A murine 3F1 VH variant wasdesigned to substitute this residue (residue 80 of SEQ ID NO: 40) with aserine residue

(QVQLQQSDAELVKPGASVKISCKASGYTFTDHAIHWVKQKPEQGLDWIGYISPGNGDIKYNEKFKDKVTLTADKSSSTASMHLNSLTSEDSAVYFCKRSLLALDYWGQGTTLTVSS; SEQ ID NO: 42).

Humanized 3F1 antibody variable domains were also designed and arepresented in the following Table. In the Table, VH or VL domains areindicated, followed by a digit to show the variant number. Domains withthe digit “0” represent the humanized sequence without anyback-mutation. All VH variants presented were designed with substitutionof the unpaired cysteine residue (residue 80 of SEQ ID NO: 40) with aserine residue or with an amino acid having a hydrophobic side chain(e.g., tyrosine).

TABLE 19. 3F1 variant variable domains mAb Chain Sequence SEQ ID NO 3F1VL0 DIQMTQSPSSLSASVGDRVTITCKASQDVGTNIAWYQQKPGKAPKLLIYSASTRHTGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCQQYSSFPLTFGQGTKVEIK 2543F1 VL1 DIQMTQSPSSLSASVGDRVTITCKASQDVGTNIAWYQQKPGKAPKVLIYSASTRHTGVPSRFSGSGSGTDFTLTISSLQPEDFA TYFCQQYSSFPLTFGQGTKVEIK 2553F1 VH0 QVQLVQSGAEVKKPGASVKVSCKASGYTFTDHAIHWVRQAPGQGLEWMGYISPGNGDIKYNEKFKDRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARSLLALDYWGQGTLVTVSS 256 3F1 VH1QVQLVQSGAEVKKPGASVKVSCKASGYTFTDHAIHWVRQAPGQGLEWMGYISPGNGDIKYNEKFKDRVTMTADKSSSTAYMQLRSLRSDDTAVYFCKRSLLALDYWGQGTLVTVSS 257 3F1 VH2QVQLVQSGAEVKKPGASVKISCKASGYTFTDHAIHWVRQAPGQGLEWIGYISPGNGDIKYNEKFKDRVTLTADKSSSTASMHLRSLRSDDTAVYFCKRSLLALDYWGQGTLVTVSS 258 3F1 VH3EVQLVQSGAEVKKPGASVKVSCKASGYTFTDHAIHWVRQAPGQGLEWMGYISPGSGDIKYNEKFKDRVTMTADKSSSTAYMQLRSLRSDDTAVYFCKRSLLALDYWGQGTLVTVSS 259 3F1 VH4EVQLVQSGAEVKKPGASVKISCKASGYTFTDHAIHWVRQAPGQGLEWIGYISPGSGDIKYNEKFKDRVTLTADKSSSTASMHLRSLRSDDTAVYFCKRSLLALDYWGQGTLVTVSS 260

Variable domain pairs were selected for initial expression of fullantibodies and testing. Among the pairs selected for 3F1 were VL0 andVH0, VL1 and VH1, VL1 and VH2, VL1 and VH3, VL1 and VH4, VL0 and VH3.

Example 8. Characterization of Humanized Antibodies

Humanized IgGl antibodies having variable domains as described in theprevious example were expressed and subjected to characterizationanalysis including flow cytometry-based binding analysis withMDA-MB-231-STn cells; binding analysis by BSM ELISA; and glycan arrayanalysis.

In flow cytometry-based binding studies, antibodies were screened over aconcentration range of 0 to 300 nM, comparing binding to MDA-MB-231cells with or without transfection-induced STn expression. Binding wasdetermined using an anti-human APC conjugated secondary antibody andonly live cells were considered (based on propidium iodide negativegating). 5,000 events were collected per sample on average. Data wereanalyzed using FlowJo software (Asland, OR) and resulting APC means and% APC were obtained. These data were log transformed then fit to anonlinear regression model to obtain a dose response curve and EC₅₀binding information. Human isotype IgGl antibody was used as an isotypenegative control. Epidermal growth factor receptor (LA22, EMD Millipore,Billerica, MA) was used as a positive control.

For BSM ELISA analysis, antibodies were screened over a concentrationrange of 0 to 100 nM on bovine submaxillary mucin (BSM) coated wells. Asubset of wells were treated with mild periodate solution beforeantibody binding to remove the side chain on terminal sialic acidresidues (destroying the STn antigen). Optical densities of periodateand non-periodate-treated wells were determined and log transformed thenfit to a nonlinear regression model to obtain a dose response curve.Optical density values obtained from periodate-treated wells weresubtracted from non-periodate treated wells to obtain aperiodate-sensitive STn binding curve and corresponding EC₅₀ values.

Glycan array analysis was carried out as described previously andantibodies were assigned array glycan binding profiles according to theparameters described therein.

Results from flow cytometry, ELISA, and glycan array analysis arepresented in the following Table.

TABLE 20 Antibody characterization results Clone ID Humanized variabledomain pair MDA-MB-231-STn cell binding [EC₅₀ (nM)] BSM ELISA [EC₅₀(nM)] Array glycan binding profile 3F1 VL1,VH1 0.3 1.8 Group 1 3F1VL1,VH2 0.7 1.4 Group 1 3F1 VL1,VH4 9.8 6.5 Group 1 3F1 VL1,VH3 20.112.2 Group 1 2G12-2B2 VL0,VH3 2.0 4.2 Group 1 2G12-2B2 VL2,VH2 0.6 2.9Group 1 2G12-2B2 VL0,VH2 0.8 1.8 Group 1 2G12-2B2 VL2,VH1 1.4 4.4 Group1 2G12-2B2 VL0,VH1 2.1 4.5 Group 1 5G2-1B3 VL1,VH2 0.1 Not DeterminedGroup 4 5G2-1B3 VL1,VH3 0.2 Not Determined Group 4 5G2-1B3 VL2,VH2 0.2Not Determined Group 4 5G2-1B3 VL2,VH1 0.3 Not Determined Group 45G2-1B3 VL1,VH1 0.1 Not Determined Group 4

All antibodies tested demonstrated binding to cell- and BSM-associatedSTn. No binding was observed with human IgGl isotype control (SouthernBiotech, Birmingham, AL). Humanized 5G2-1B3 binding was not periodatesensitive in ELISA assays, so a reliable EC₅₀ could not be determined byBSM ELISA.

Based on the results of characterization experiments, two antibodiesfrom each clone group were selected for one liter expression andresulting antibodies were tested again according to the same procedures(see results presented in the following Table).

TABLE 21 Antibody characterization results after one liter productionClone ID Humanized variable domain pair MDA-MB-231-STn cell binding[EC₅₀ (nM)] BSM ELISA [EC₅₀(nM)] 3F1 VL1,VH1 0.48 0.86 3F1 VL1,VH2 0.670.57 2G12-2B2 VL0,VH3 1.20 0.80 2G12-2B2 VL2,VH2 0.45 1.82 5G2-1B3VL1,VH2 0.34 Not Determined 5G2-1B3 VL1,VH3 1.57 Not Determined

All antibodies expressed demonstrated an EC₅₀ of less than 2 nM for bothcell-associated and BSM-associated STn binding.

Example 9. Analysis of Humanized Antibodies With Antibody-DrugConjugates

Antibody-drug conjugate (ADC) versions of humanized antibodies describedin the previous example were developed by conjugation with monomethylauristatin E (MMAE). This was carried out by contacting antibodies withmaleimidocaproyl-valine-citruline-p-aminobenzyloxycarbonyl-monomethylauristatin E (MC-γc-PAB-MMAE, referred to herein as CL-MMAE). Theresulting conjugation is maleimide-cysteine based, where the antibodyinterchain disulfide bonds are reduced with TCEP and then linked to themaleimide moiety of the drug.

Conjugated antibodies were desalted on Sephadex G50 columns to removeresidual unreactive toxins and then dialyzed in 30 mM HEPES pH 7.7 with150 mM NaCl.

ADC antibodies were then assessed in an ADC cytotoxicity assay usingMDA-MB-231 cells (parental or transfected for enhanced expression ofSTn). Parental cells were grown in Eagle’s Minimum Essential Medium(EMEM) supplemented with 10% FBS, 1x Pen/Strep and 45 µg/mL gentamycin.STn positive cells were grown in the same media except with the additionof 1 mg/mL G418 for antibiotic selection. Cells were seeded separately(4,000 cells/well for parental cells or 2,000/well for STn positivecells) in 96 well plates using proper media described above. Cells weregrown overnight. After 16-20 hours, cells were treated with varyingconcentratinos of test antibodies in triplicate (50 nM to 0.012 nM) for72 hours. Then, cells were analyzed using an ADC CELLTITER-GLO®luminescent cell viability assay kit (Promega, Madison, WI) to determinethe amount of ATP present, an indicator of metabolically active cells.The assay uses a single reagent that is added directly to the culturedcells in serum-supplemented medium. The reagent lyses the cells andgenerates a luminescent signal proportional to the amount of ATPpresent. Luminescent signals were analyzed and used to calculate IC₅₀values for each antibody used based on their ability to kill STnpositive cells (see the following Table).

TABLE 22 IC₅₀ values for humanized ADC antibodies Humanized AntibodyIC₅₀ (nM) 3F1, VL1,VH1 1.30 3F1, VL1,VH2 1.04 5G12-1B3, VL1,VH2 2.585G12-1B3, VL1,VH3 7.89 2G12-2B2, VL0,VH3 7.55 2G12-2B2, VL2,VH2 5.17

All antibodies tested demonstrated IC₅₀ values in the single nanomolarrange indicating a strong capability for each to kill STn expressingcells.

Example 10. MDA-MB-231 Xenograft Model Studies

Xenograft model studies are carried out to test humanized ADC antibodiesin vivo. Tumors are induced in mice through subcutaneous injection ofcancerous cells. Cancerous cells used for injection are selected from:(1) cells transfected to induce expression of STn (MDA-MB-231 STn+cells), (2) cancer cell lines that naturally express STn on theirsurface, and (3) patient-derived tumor cells, taken from primary humanpatient tumors.

Models using patient tumor cells may more faithfully replicate humantumor biology and better predict drug response than other models. Insome experiments, patient tumor cells are derived from colorectal cancerpatients. In some experiments, patient tumor cells are selected aftersearching RNA sequence databases to identify cells expressing ST6GalNAcI. In some experiments, patient tumor cells are selected based onexpression of STn, as assessed by immunostaining or flow cytometryanalysis using anti-STn antibodies.

Once tumor cells are injected into study mice, tumors are allowed todevelop until a desired tumor volume (typically between about 175 mm³ toabout 225 mm³) is reached. At this point, mice are segregated intotreatment groups. Mice are then treated with compositions that includehumanized MMAE-conjugated antibodies, irrelevant control antibodies ornaked (non-conjugated) antibody controls. Doses are sufficient todeliver from about 1 to about 20 mg of antibody per kilogram of mousebody weight. Mice receive either a single dose or multiple doses (e.g.,once per week for three weeks). During treatment, mice are monitored forchanges in weight and tumor volume. Tumor volumes in mice treated withhumanized ADC antibodies are reduced by about 20 to 100%.

Example 11. Tissue Studies

Humanized antibodies are directly labeled with biotin or pre-complexedwith anti-human IgG biotin labeled secondary antibodies. Formalin-fixedparaffin embedded tissue microarray tissue sections are de-paraffinzed,rehydrated, and subjected to antigen retrieval before treatment withbiotinylated antibodies or antibody complexes. Antibody binding to thetissues is detected using the VECTASTAIN™ ABC kit (Vector Laboratories,Burlingame, CA) to produce a visual precipitate. Followingcounterstaining with hematoxylin, slides are scored microscopically in ablinded fashion for staining intensity, frequency, and localization. Foreach candidate antibody, normal tissue microarrays (AC1, Super BioChips, Seoul, Korea) containing 60 samples (2 human donors each for 30organs or subregions of organs) are used to assess normal tissuebinding. Human cancer tissue microarrays (MA2 and MA4, Super Bio Chips,Seoul, Korea) containing 118 donor tumor samples are tested to assessantibody binding to cancerous cells. A total of 13 different commontumor types are tested overall, with numerous subclassificationannotations captured for each tumor type. Humanized antibodies bind tocancerous cells (including pancreatic and colorectal cancer cells) inhuman cancer tissue sections with minimal or no binding to cells innormal tissue sections.

Example 12. In Vitro Viability Assays

Experiments are carried out to identify cancer cell lines (e.g.,pancreatic and colorectal cell lines) that express STn intrinsically.Among those tested are colorectal cancer cell lines [e.g., LS180(CL-187), COLO205 (CL-222), TB4 (CCL-248), HT29 (HTB-38), RKO(CRL-2577), SW480 (CCL-228) and SNU-C2A (CCL-250.1) cell lines] andpancreatic cell lines [e.g., Panc-1 (CRL-1469), CFPAC1 (CRL-1918), HPAC(CRL-2119), ASPC1 (CRL-1682), BXPC3 (CRL-1687), CPAN1 (HTB-79), andHPAFII (CRL-1997) cell lines].

Flow cytometry is utilized to assess STn expression. Anti-STn antibodiesare combined with cells from the cell line being tested. Binding isdetermined using an APC conjugated secondary antibody and only livecells are considered (dead cells filtered out using a propidium iodidenegative gate). 5,000 events are collected per sample on average. Dataare analyzed using FlowJo software (Ashland, OR) and resulting APC meansand % APC are obtained. Normal IgGl antibody is utilized as an isotypecontrol. Cell lines are identified that express STn. Flow cytometry isrepeated with humanized antibodies using cell lines found to expressSTn. Humanized antibodies are found to bind to STn-expressing cancercell lines.

Cell viability studies are carried out with STn-expressing cancer celllines identified. Humanized antibodies are used to form ADC antibodiesconjugated with MMAE. Cells are treated with the humanized ADCantibodies and IC₅₀ values are calculated for each cell line. HumanizedADC antibodies tested are effective in killing STn-expressing cancercells lines tested.

Example 13. Tissue Cross-Reactivity Study

Tissue cross-reactivity (TCR) studies are carried out to assess thebinding profile (both on- and potential off-target binding) ofantibodies to human and relevant species used in nonclinical safetytesting. For initial characterization and optimization, a preliminaryTCR study is conducted to assess the staining pattern of the humanizedlead ADC antibodies in human tissues (normal versus cancerous). Leadcandidates demonstrate an optimal staining profile with specific cancercell staining and no or minimal staining of normal tissues.

Cryosections of human, mouse, rat, and cynomolgus monkey normal tissuepanel (e.g., brain, colon, heart, liver, lung, pancreas, smallintestine, spleen, and stomach) are probed for anti-STn antibodybinding. Carcinoma cells within human pancreatic neoplasm are used aspositive control tissues and stromal cells within the same tissue areused as negative controls. Detection utilizes an indirectimmunoperoxidase technique followed by an ABC tertiary system where theanti-STn humanized antibodies are pre-complexed with biotinylatedsecondary antibody before tissue incubation. Validation staining runsare undertaken with a limited panel of tissues to determine properantibody concentrations and conditions before staining the entire tissuepanel.

Example 14. Toxicology and Pharmacokinetic Studies

Toxicology studies are carried out in rats using humanized ADCantibodies to identify antibodies with toxic effects and to determineno-observed-adverse-effect-level (NOAEL) for each antibody. Rats are asuitable model because mice are resistant to auristatin, the cytotoxiccomponent of the ADC antibodies. Both single dose and multiple dosestudies are undertaken using either 1 mg/kg, 2.5 mg/kg, or 5 mg/kgdoses. Multiple rats are included in each treatment group. For singledose studies, animal health and body weights are monitored and rats aresacrificed at different time points after intraperitoneal (IP) antibodyadministration, including 72 hours after treatment and 2 weeks aftertreatment. For multiple dose studies, rats receive IP antibodyinjections at day 0, at week 2 and at week 4. In these studies, rathealth and body weights are monitored and rats are sacrificed atdifferent time points after administration, including 24 hours after thelast dose and 2 weeks after the last dose.

After sacrifice, organs (adrenal gland, brain, colon, intestine, heart,kidney, lung, mandibular salivary gland, pancrease, spleen, stomach, andthyroid gland) are harvested, formalin-fixed and paraffin-embedded forhematoxylin and eosin (H&E) staining and pathological evaluation.

Rats subjected to single and multiple dose administration of humanizedADC antibodies tested do not show any signs of weight loss or adversehealth effects. Organs also appear normal at all time points tested.

In rats utilized for pharmacokinetic analysis, blood samples areobtained before and throughout the study period to quantify serumconcentration levels of study antibodies and to conduct pharmacokineticmodeling. Blood is obtained at least 24 hours prior to dosing, at day 1(approximately 1, 4, and 8 hours post-dose), at day 2 (at approximately24 hours post-dose), at day 3 (at approximately 48 hours post-dose), atday 4 (at approximately 72 hours post-dose), and at various timespost-dose based on single dose results and multiple dose study designs.

Blood samples are allowed to clot and the sera is separated bycentrifugation. Resulting samples are subjected to clinical pathologicalevaluations (clinical chemistry, hematology, and coagulation). Clinicalchemistry evaluation includes analysis of sodium creatinine, totalprotein, potassium, alkaline phosphatase, triglycerides, chloride,alanine aminotransferase, total bilirubin, calcium aspartateaminotransferase, albumin, inorganic phosphorus, glucose, globulin,urea, nitrogen, cholesterol, and albumin/globulin ratio. Hematologyevaluation includes evaluation of hematocrit, mean corpuscularhemoglobin concentration, hemoglobin, reticulocyte count (absolute andrelative), platelet count, erythrocyte count, mean platelet volume,total white blood cell count, mean corpuscular hemoglobin, differentialwhite blood cell count (absolute & relative), mean corpuscular volume,and red blood cell distribution width. For coagulation analysis,prothrombin time and activated partial thromboplastin time aredetermined. Clinical pathological evaluations indicate no adverseeffects from treatment with humanized ADC antibodies.

Example 15. Evaluation of Patient-Derived Tumor Cells

Experiments are conducted to characterize the STn expression profile ofpatient-derived xenograft (PDX) cells. Patient-derived cancer cells areused to generate tumors in NOD/SCID mice as described previously. Cellsfrom resulting PDX tumors are removed, dissociated and screened for STnexpression. Screening is carried out initially by immunohistochemistry(IHC), then confirmed by flow cytometry analysis. Cells from PDX tumorswith the best expression of STn are selected for continued studies.

In one continued study, cells from the selected PDX tumors are culturedin vitro. Some cultures are treated with humanized anti-STn antibodies,described herein, that are conjugated with a cytotoxic agent, MMAE, toform antibody-drug conjugates (ADCs). The ability of these ADCs to killthe cultured cells is determined using cell viability assays. Studiesare carried out to compare treatment of these cultures with or withoutchemotherapeutic agents. Humanized anti-STn ADCs are able to kill cellsfrom PDX tumors that express STn. When cells are first treated withchemotherapeutic agents, the ability of humanized anti-STn ADCs to killthese cells is enhanced.

In another continued study, cells from the selected PDX tumors arecultured in vitro and treated with or without chemotherapeutic agents.STn expression before and after chemotherapeutic agent treatment isevaluated. STn expression in the cells evaluated is increased afterchemotherapeutic agent treatment.

Example 16. Antibody Testing Using OVCAR3 Xenograft Model

The effectiveness of humanized anti-STn antibodies to reduce cancercells in an in vivo tumor model is evaluated. NOD/SCID mice are injectedwith 5 × 10⁵ OVCAR3 cells in a MATRIGEL® (Corning Life Sciences,Corning, NY) suspension to induce OVCAR3 tumor formation. Once miceexhibit tumor volumes ranging from 175-225 mm³, they are randomized intogroups with essentially equivalent group mean tumor volumes. Humanizedanti-STn antibodies with MMAE conjugates, isotype control antibodies, orvehicle control [20 mM citrate (pH 5.5) and 150 mM NaCl] areadministered at a dose of 2.5 mg/kg and mice are monitored for changesin tumor volume and body weight twice weekly for 4 weeks after treatment(or until tumor size reaches an endpoint volume of ≥ 1000 mm³). Tumorsare then extracted and evaluated for the presence of viable tumor cellsand STn expression. Antibodies capable of inhibiting or reducing tumorvolume; reducing cancer cell numbers; and/or STn expression in tumorsare identified and used in further studies.

Example 17. Evaluation of PDX Samples After Single Antibody Treatment

Experiments are carried out to compare responsiveness of PDX models withdiffering characteristics to anti-STn antibody therapy at differentantibody doses. Slow frozen tissue from a passaged ovarian carcinoma PDXtumor is implanted into NOD/SCID mice to generate PDX tumors in thosemice over 16 weeks. Tumors are harvested and reinjected into 25 NOD/SCIDmice to generate PDX tumors over 12 weeks. Resulting tumors are againharvested and reinjected into 52 NOD/SCID mice and tumors are allowed toform for 12 weeks. These mice are then treated with intraperitonealinjections of humanized anti-STn antibodies (conjugated with MMAE) at2.5 mg/kg or 5 mg/kg doses; isotype control antibody; or vehicle control[20 mM citrate (pH 5.5) and 150 mM NaCl]. Changes in mouse weight andtumor volume are monitored twice weekly after treatment (or until tumorsize reaches an endpoint volume of ≥ 1000 mm³). Tumors are thenextracted and evaluated using flow cytometry for tumor cell viabilityand STn expression. PDX tumors responsive to anti-STn antibody treatmentare identified.

Example 18. Multi-dose Treatment of PDX Tumors

Cells from PDX tumors with demonstrated responsiveness to humanizedanti-STn treatment are selected for use in a multi-dose antibodytreatment study. Slow frozen tissue from a passaged ovarian carcinomaPDX tumor is implanted into NOD/SCID mice to generate PDX tumors inthose mice over 16 weeks. Tumors are harvested and reinjected into 25NOD/SCID mice to generate PDX tumors over 12 weeks. Resulting tumors areagain harvested and reinjected into 52 NOD/SCID mice and tumors areallowed to form for 12 weeks. These mice are then treated weekly, for 4weeks, with intraperitoneal injections of humanized anti-STn antibodies(conjugated with MMAE) at a dose of 5 mg/kg; isotype control antibody;or vehicle control [20 mM citrate (pH 5.5) and 150 mM NaCl]. Changes inmouse weight and tumor volume are monitored twice weekly after treatment(or until tumor size reaches an endpoint volume of ≥ 1000 mm³). Tumorsare then extracted and evaluated using flow cytometry for tumor cellviability and STn expression. PDX tumors responsive to anti-STn antibodytreatment are identified. MMAE-conjugated humanized anti-STn antibodiesare most effective at reducing tumor volume.

Example 19. Cross-Reactivity, Toxicology

Cross-reactivity studies are carried out to determine cross-reactivityof humanized anti-STn antibodies between human, cyno, and rat subjectsby immunohistochemical staining using a tissue panel. Humanized anti-STnantibodies are found to cross react with both cyno and rat subjects.Further toxicological studies are carried out in rats to assess toxicityof humanized anti-STn antibodies. Assessments include in lifeassessments such as mortality/morbidity, clinical observations, bodyweight, food consumption, body temperature, local irritation, andophthalmology. Humanized anti-STn antibodies are not found to be toxicat doses of 10 mg/kg and under.

Example 20. Pharmacokinetic Studies

Humanized anti-STn antibodies conjugated with MMAE are administered torodent (e.g., rat) or primate study models at a dose of 2.5 mg/kg or 5mg/kg to evaluate antibody half-life and clinical pathology (e.g.,clinical chemistry, hematology, and coagulation). Assessments are madeat 72 hour, 2 week and 4 week time points.

For half-life analysis, antibody body fluid concentrations aredetermined after 1 hour, after 4 hours, after 8 hours, after 24 hours,after 48 hours and after 72 hours from antibody administration.

For clinical pathology, blood samples are collected from study subjectsprior to dosing (pretest), and at multiple time points after dosing. Forclinical chemistry, sodium creatine, total protein, potassium, alkalinephosphatase, triglycerides, chloride, alanine aminotransferase, totalbilirubin, calcium aspartate aminotransferase, albumin, inorganicphosphorus, glucose, globulin, urea, nitrogen, cholesterol, andalbumin/globulin ratio are measured. For hematology, hematocrit, meancorpuscular hemoglobin concentration, hemoglobin, reticulocyte count(absolute and relative), platelet count, erythrocyte count, meanplatelet volume, total white blood cell count, mean corpuscularhemoglobin, differential white blood cell count (absolute and relative),mean corpuscular volume, and red blood cell distribution width aredetermined. For coagulation, prothrombin time and activated partialthromboplastin time are evaluated.

Finally, study animals are euthanized and organs (adrenal gland, brain,colon, intestine, heart, kidney, lung, mandibular salivary gland,pancreas, spleen, stomach and thyroid gland) are harvested,formalin-fixed and paraffin-embedded for H&E staining and pathologicalevaluation by a board-certified pathologist. No adverse effects areobserved with humanized antibodies tested.

Example 21. Stable Cell Line Producing Humanized Anti-STn Antibody

Stable cells lines suitable for transition to a GMP facility forproduction are generated to produce humanized anti-STn antibodies.FREEDOM® pCHO 1.0 vectors (Thermo Fisher Scientific, Waltham, MA) areused to generate constructs expressing humanized antibodies having oneor more of the variable domains presented in herein. Constructs areintroduced by transfection into Chinese Hamster Ovary (CHO) suspensioncells using the Gibco FREEDOM® CHO-S® kit (Thermo Fisher Scientific,Waltham, MA) and cells are cultured according to kit instructions toselect puromycin-resistant cells exhibiting stable expression of theintegrated constructs. Resulting stable cell lines are grown forantibody production and storage.

Example 22. Generation of SKOV3 Cell Lines With Enhanced ST6GalNAc IExpression

SKOV3 cells were transduced with lentiviral vectors delivering ST6GalNAcI expression constructs (hST6GalNAc I_pRc-CMV). Stable cell pools weregenerated and 6 clones with varying expression of ST6GalNAc I [asdetermined by quantitative polymerase chain reaction (qPCR) analysis]were selected (see the following Table).

TABLE 23 Expression levels of ST6GalNAc I in selected clones Clone IDST6GalNAc mRNA expression level (fold expression level over control)Clone 7 165 Clone 8 105 Clone 10 15 Clone 13 125 Clone 15 20 Clone 16 30

Clones 7, 8, and 13 demonstrated the highest level of ST6GalNAc I mRNAwhen compared to levels in non-transduced cell lines.

Example 23. Xenograft Tumor Model Studies Using Cells With Varying STnExpression Levels

Experiments are carried out to compare responsiveness of xenografttumors with varying levels of STn expression to humanized anti-STnantibody treatment. Tumor cells with varying levels of STn expression(i.e., cells with no STn expression, cells with low levels of STnexpression, cells with intermediate levels of STn expression, and cellswith high levels of STn expression) are obtained. These include cellsthat have been modified to over-express ST6GAlNAC I; cells withknockdown of ST6GalNAc I expression; and non-modified cells that have noSTn expression, low expression level, intermediate expression level, orhigh expression level. The tumor cells are implanted into NOD/SCID miceto generate tumors.

Mice are then treated with intraperitoneal injections of humanizedanti-STn antibodies (with or without conjugated MMAE); isotype controlantibody; or vehicle control [20 mM citrate (pH 5.5) and 150 mM NaCl]for 8 weeks. Changes in mouse weight and tumor volume are monitoredtwice weekly after treatment. After 8 weeks, mice receiving anti-STnantibody treatments are randomized to either continue 8 more weeks ofthe initial therapy or to be treated with vehicle control for 8 weeks.At the end of the 16-week period, serum samples are obtained and tumorsare extracted for evaluation using flow cytometry for tumor cellviability and STn expression.

Anti-STn antibodies conjugated with MMAE yield the highest level ofanti-tumor activity. Discontinuation of anti-STn-MMAE treatment uponrandomization promotes tumor resurgence while prolonged therapy withanti-STn-MMAE antibodies prevents tumor resurgence.

Example 24. Screening of Cell Lines for Expression of STn

Breast, colon, ovary, lymphocyte, bone marrow, gastric, paneratic,colorectal, skin cell and other oncological indication cell lines arescreened for STn expression. Cell lines tested include SNU-16 cells,LS-174T cells, MC38 cells, COLO205, RKO, HT29, Panc1, HPAC, HPAFII,TOV-112D cells, TOV-21G cells, Jurkate E6.1 cells, K-562 cells, B16-F0cells, and B16-F10 cells.

Colorectal cell lines [for example, LS180 (CL-187), COLO205 (CL-222),TB4 (CCL-248), HT29 (HTB-38), RKO (CRL-2577), SW480 (CCL-228), andSNU-C2A (CCL-250.1)] are selected for screening based upon ST6GalNAcIexpression and desirable characteristics (e.g., doubling time,tumorigenic properties, chemo-resistance and antigen expression). STnexpression is tested on both cells grown in vitro and in vivo given thatsurface and enzymatic expression may be different based on cell growthconditions during development and differentiation.

Cell lines are subjected to STn expression analysis by flow cytometry.Humanized anti-STn antibodies are used to probe for STn expression and ahuman isotype control is utilized as a negative control.

Each cell line is propagated in culture and distributed among differentgrowth formats. For in vivo formats, cells are injected into NOD/SCIDmice in a MATRIGEL™ (Corning Life Sciences, Corning, NY) suspension [5 ×10⁶ cells at a ratio of 1:1 (v/v) with MATRIGEL™] to generate axenograft model. Mice with mean tumor volumes of 200 mm³, 400 mm³, 600mm³, or 1000 mm³ are sacrificed and tumors are extracted for STnexpression analysis by flow cytometry and for formalin-fixed paraffinembedding for immunohistochemical analysis.

Cells demonstrating STn expression are used for further studiesincluding selection, characterization, and testing of anti-STnantibodies. Some cells demonstrating low or no STn expression aretransfected to express STn before use in further studies (e.g.,selection, characterization, and testing of anti-STn antibodies).

Example 25. Testing Humanized Anti-STn Antibodies in STn-ExpressingColorectal Cell Lines

Humanized anti-STn antibodies are assessed for their capacity for beinginternalized into STn-expressing colorectal cell lines. Anti-CEAantibodies are used as a positive control. CEA is known to be expressedon the surface of many types of colon cancer cells and may beinternalized in colorectal cells, along with other cell types expressingCEA. Anti-STn antibodies as well as controls are covalently labeled withALEXA FLUOR™ 488 (Thermo Fisher, Waltham, MA) according tomanufacturer’s directions. Surface bound antibody signal is blockedusing anti-ALEXA FLUOR™ 488 antibody before assessing internalizationvia flow cytometry. Results indicate that anti-STn antibodies areinternalized by STn-expressing colorectal cell lines.

Example 26. Bystander Killing Assay

STn-positive cells are seeded into a transwell where STn-low or-negative expressors are seeded onto the bottom of the plate. Wells withonly STn-positive or only STn-negative cells are included as controls.Doses of 0 to 300 nM of anti-STn antibodies with MMAE conjugates areadded to the cultures (as well as free MMAE in some wells as a toxiccontrol) and viability of the STn-low or -negative expressing cells isdetermined using the Promega (Madison, WI) ADC CELLTITER-GLO®luminescent cell viability assay kit, which determines the amount of ATPpresent as an indicator of metabolically active cells.

Results indicate that STn expressing cells internalize the anti-STnMMAE-conjugated antibodies. The dying cells release cleaved free MMAEwhich migrates across the transwell membrane and little to no bystanderkilling is observed by way of toxicity in the non/low-STn expressingcells.

Example 27. Plasma Stability Study

The plasma stability of humanized anti-STn antibodies is evaluated inhuman, cynomolgus monkey, rat and mouse plasmas. Antibodies are spikedinto human, cynomolgus monkey, rat and mouse plasmas in vitro and thenincubated at 37° C. for up to 14 days. The concentrations of totalhumanized antibodies, humanized antibody MMAE conjugtes, and free MMAEin the plasma samples are quantified at different days using immunassaysand LC-MS-based methods. The drug antibody ratio (DAR) is also assessedin the same samples. Antibodies remain relatively stable in plasma andDAR demonstrate little variation over the course of the study.

Example 28. Identification of STn Containing Proteins Using AntibodyMicroarrays

Cancer cells (MDA MB 231) with or without transfection to induce STnexpression were used to identify proteins carrying STn glycosylation.Crude cell lysate from MDA-MB-231 STn+/- were probed with printedantibody microarrays (Rho et. Al, 2013). Each array containsapproximately 3500 human-protein specific antibodies, targetingapproximately 2100 unique proteins, in triplicate, that are covalentlyimmobilized via N-hydroxysuccinimide (NHS)-ester reactive 3-D thin filmsurface slides (Nexterion H slide, Schott). Targets of printedantibodies were selected from proteins related to cancer, signalingproteins, and previously identified plasma cancer proteins.

Frozen microarray slides were equilibrated to room temperature for 30minutes and hydrated in 0.5% Tween20 in phosphate buffered saline (PBS)and then rinsed with distilled/deionized water (dd H₂O). The slides werethen blocked by incubation for 30 min with 0.3% (v/v) ethanolamine in 50mM sodium borate, pH 8, followed by 30 min with 1% BSA (w/v), 0.5% Tween20 in PBS. Next, the arrays were washed with 0.5% Tween 20 in PBS,followed by dd H₂O. Then, the arrays were dried by centrifugation at 500rpm for 8 min in a swinging bucket rotor with a slide rack holder(Sorvall Legend RT). The antibody-printed area of the arrays was coveredwith a coverslip (mSeries Lifter Slips, 22x25x1 mm, Thermo Scientific).

To detect the presence of STn containing proteins, STn+/- cells werecultured and crude cell lysate was collected in 3 biological replicatesto obtain samples (N=3 for STn+ and N=3 for STn-). Lysate was pipettedonto the slide at the microarray/coverslip junction and incubated for 60min at room temperature. The slides were then washed two times for 5 minwith 0.5% Tween 20 in PBS. STn containing glycoproteins were detectedafter incubation with Siamab’s STn antibodies (Hu3F1,L1H1;Hu2G12-2B2,L2H2; Hu5G2-1B3,L1H2 and Hu3F1,L1H1) conjugated tofluorescent Cyanine5 dye. The arrays were washed two times for 5 minwith 0.5% Tween 20 in PBS, followed by two times with PBS (5 min each)and once with dd H₂O water followed by drying by centrifugation. Todetermine background levels of signal, the arrays were incubated withjust STn antibody (no cell lysate added) and the resulting signals wereused for background subtraction. The slides were then scanned on aGenePix 4200A microarray scanner (Axon Instruments) to produce red (Cy5)images. Spot fluorescent intensities of the scanned array images wereobtained using Genepix Pro 6.0 image analysis software.

Differences in Fluorescence intensity (FI) between STn + and STn-conditions was analyzed by 3 statistical methods: (1) Effect size(((Mean FI STn+ cells - Mean FI STn-cells))/Standard deviation of STn-cells). An effect size >3 is considered desirable in this assay. (2)p-value. A p value <0.25 is desirable in this assay. (3) Ratio(2^((Log mean FI STn+ cells) -) ^(Log mean FI STn- cells)). A ratio >1.2 indicates a protein is increased in STn+ cells and a ratio <0.8suggests a protein is decreased in STn+ cells.

Each antibody was seen to have different binding properties, butconfirmation of certain protein binding between the different antibodieswas strong proof of overall upregulation of Sialyl Tn content in cancer.Additionally, known STn carriers MUC16 and MUC1 were detected in thisassay. The top 35 hits consisted of proteins located in: plasma membrane(7), extracellular space (8), nucleus (6) and cytoplasm (17). Overall,Hu3F1,L1H1 had the broadest specificity, detecting upregulation in 63 ofthe total 86 proteins detected. An example of some proteins with STnglycosylation detected using Hu3F1,L1H1, Hu2G12-2B2,L2H2, Hu5G2-1B3,LIH2in this assay are listed in table below.

TABLE 24 Proteins with increased STn glycosylation Gene Cellularlocation Hu3F1 L1H1 p value Hu3F1 L1H1 effect size Hu2G12-2B2 L2H2 pvalue Hu2G12-2B2 L2H2 effect size Hu5G2-1B3 L1H2 p value Hu5G2-1B3 L1H2effect size IL10 Extracellular Space 0.01 4.11 0.01 6.90 0.00 2.23 SPP1Extracellular Space 0.03 13.76 0.10 2.78 N/D N/D LY6D Plasma Membrane0.32 2.63 0.24 1.96 0.02 13.19 MUC16 Cell membrane, secreted 0.04 2.64N/D N/D 0.07 79.10 F5 Secreted, Plasma Membrane 0.02 5.69 0.04 2.89 0.201.59 PDPK1 Cell membrane-peripheral membrane protein, Cytoplasm 0.0812.38 0.40 1.36 0.03 7.05 Ihh Extracellular space 0.15 24.84 0.54 0.930.83 0.07 IHH Extracellular Space 0.35 1.32 0.05 3.95 N/D N/D IHHExtracellular Space N/D N/D N/D N/D 0.05 12.07 SMS Cytoplasm 0.10 3.110.13 30.82 0.03 7.22 MAPK3 Cytoplasm 0.01 6.73 0.11 3.07 0.04 11.96 OAS1Cytoplasm 0.01 4.69 0.05 32.20 0.89 0.28 CRADD Cytoplasm 0.13 1.26 0.171.68 0.06 28.19 GRB2 Cytoplasm 0.01 5.38 0.14 6.83 0.07 7.23 PRDX6Cytoplasm 0.06 2.48 0.25 9.99 0.19 2.35 PCNA Nucleus 0.05 2.95 0.19 2.790.11 21.73 CUX1 Nucleus 0.06 2.35 0.00 6.74 N/D N/D

All proteins listed showed affinity for at least one STn antibodysuggesting the presence of STn glycosylation. IHH appears on the list asthree separate entries. These are captured by three unique antibodies toIHH. The following proteins showed binding with two different antibodyclones: IL10, SPP1, LY6D, MUC16, F5, PDPK1, SMS, MAPK3, OAS1, CRADD,GRB2, PRDX6, PCNA and CUX1, strongly demonstrating their STnglycosylation.

Among these IL10, SPP1, LY6D and MUC16 have extracellular or cellmembrane localization and have been previously implicated as cancerbiomarkers.

IL10: Interleukin-10 inhibits the synthesis of a number of cytokines,including IFN-gamma, IL-2, IL-3, TNF and GM-CSF that are produced byactivated macrophages and by helper T-cells.

SPP1: Osteopontin is activated by ligand, sialic acid, and is essentialfor Type I immunity. It can interact with CD44. SPP1 acts as a cytokineand enhances production of interferon-gamma and interleukin-12 anddecreases production of interleukin-10.

MUC16: Mucin-16 or CA-125, is a known cancer biomarker for ovariancancer.

LY6D: Lymphocyte antigen 6D acts as a B cell specification marker at thespecification stage of lymphocytes between B- and T-cell development.

Some of the proteins identified by the screen were unique to only oneSTn antibody clone. Proteins showing glycosylation with Hu3F1,L1H1 arerepresented in the table below.

TABLE 25 Proteins recognized by Hu3F1,L1H1 Gene Location Hu3F1 L1H1 pvalue Hu3F1 L1H1 effect size TLN1 Plasma Membrane/Cytoplasmic side 0.0625.27 MUC1 Plasma Membrane 0.03 9.00 LIMK2 Cytoplasm 0.02 8.78 MAPRE1Cytoplasm 0.03 7.91

TLN1: Talin-1 is a part of the connection between cytoskeletalstructures and plasma membrane. TLN1 was previously identified in mouseinsertional mutagenesis experiments suggesting a causal role in cancer.TLN1 expression correlates with invasion and migration of cancer cells.

MUC1: The beta subunit of Mucin-1 contains a C-terminal domain which isinvolved in cell signaling, through phosphorylation and protein-proteininteractions, through which it can promote tumor growth. In B cells,Muc1 modulates ERK, SRC and NF-Kappa-B signaling pathways. While inactivated T-cells, it modulates the Ras/MAPK pathway.

Proteins showing glycosylation with Hu2G12-2B2,L2H2 are represented inthe table below.

TABLE 26 Proteins recognized by 2G12-2B2,L2H2 Gene LocationHu2G12-2B2,L2H2 p value Hu2G12-2B2,L2H2 effect size ALDH1A1 Cytoplasm0.01 12.70 ANO1 Plasma Membrane 0.02 6.67

ALDH1A1: Retinal dehydrogenase is a cancer stem cell marker. It has alsobeen implicated in chemoresistance.

ANO1: Anoctamin-1 is a calcium-activated chloride channel which plays arole in trans epithelial anion transport. ANO1 is amplified and highlyexpressed in breast cancer cell lines and primary tumors.

Proteins showing glycosylation with Hu5G2-1B3,L1H2 are represented inthe table below.

TABLE 27 Proteins recognized by Hu5G2-1B3,L1H2 Gene LocationHu5G2-1B3,L1H2 p value Hu5G2-1B3,L1H2 effect size GPC3 Plasma Membrane0.026 8.69 HAPLN1 Extracellular Space 0.003 5.53

GPC3: Glypican-3 is a cell surface proteoglycan that bears heparansulfate. It is involved in the suppression of growth in thepredominantly mesodermal tissues and organs. An anti-GPC3 monoclonalantibody has been shown to have anti-cancer activity in mice.

Proteins showing STn glycosylation with Hu3F1,L1H1 antibody werecompared with proteins showing glycosylation using the Mu3F1. Resultsare presented in the following Table.

TABLE 28 3F1 Antibody comparison Gene Location Hu3F1,L1H1 p valueHu3F1,L1H1 effect size Ratio Mu3F1 COL4A3 Extracellular Space 0.06222.63 1.09 CCR5 Plasma Membrane 0.104 20.55 1.17 SLC30A8 Cytoplasmvesicle and cell membrane protein 0.006 8.26 1.03 CNN1 Cytoskeleton0.015 6.42 1.06 ITSN1 Endomembrane system 0.007 5.74 1.04 PKM2Cytoplasm, plasma membrane, and extracellular space 0.348 6.59 1.06LAMB3 Extracellular Space 0.116 5.28 1.12 F5 Secreted and PlasmaMembrane 0.022 5.69 1.14 MUC1 Plasma Membrane 0.028 9.00 1.00 TK1Cytoplasm 0.032 7.59 1.07 SMS Cytoplasm 0.098 3.11 1.04 PRDX6 Cytoplasm0.060 2.48 1.05 CUX1 Nucleus 0.057 2.35 1.07 MEF2C Nucleus 0.015 8.921.04 CCNE2 Nucleus 0.001 10.30 1.05 PCNA Nucleus 0.050 2.95 1.03

Example 29. Humanized Antibody Testing Using Alternative Glycan Array

All proteins listed showed affinity for both STn antibodies, indicatingthe presence of STn glycosylation. Among these COL4A3, CCR5, and MUC1have extracellular or cell membrane localization and have beenpreviously implicated as cancer biomarkers.

COL4A3: Collagen alpha-3(IV) chain. Type IV collagen is the majorstructural component of glomerular basement membranes (GBM), forming a‘chicken-wire’ meshwork together with laminins, proteoglycans andentactin/nidogen. Tumstatin, a cleavage fragment corresponding to thecollagen alpha 3(IV) NC1 domain, possesses both anti-angiogenic andanti-tumor cell activity

CCR5: C-C chemokine receptor type 5 is a receptor for a number ofinflammatory CC-chemokines. CCR5 has been implicated in the recruitmentof T-reglulatory cells (Treg) from blood into tumor sites in humancolorectal cancer. Tumor growth is delayed in CCR5-/- mice andassociated with reduced tumor Treg infiltration.

Alternative glycan arrays with 13 chemically synthesized andwell-defined glycans were also utilized to test antibody affinity andspecificity for multiple glycans in a single experiment. The alternativeglycan array includes Neu5Ac and Neu5Gc glycan pairs listed in thefollowing table.

TABLE 29 Array glycans in alternative array Glycan ID No. Glycan 1Neu5Acα6GalNAcαO(CH2)2CH2NH2 2 Neu5Gcα6GalNAcαO(CH2)2CH2NH2 3Neu5Acα6Galβ4GlcNAcβO(CH2)2CH2NH2 4 Neu5Gcα6Galβ4GlcNAcβO(CH2)2CH2NH2 5Neu5Acα6Galβ4GlcβO(CH2)2CH2NH2 6 Νeu5Gcα6Galβ4GlcβO(CΗ2)2CΗ2ΝΗ2 7Neu5Acα6GalβO(CH2)2CH2NH2 8 Νeu5Gcα6GalβO(CΗ2)2CΗ2ΝΗ2 9GalNAcαO(CH2)2CH2NH2 10 Galβ3GalNAcβO(CH2)2CH2NH2 11Gal3βGalNAcαO(CH2)2CH2NH2 12 Neu5Acα3Galβ1-3GalNAcαO(CH2)2CH2NH2 13Neu5Gcα3Galβ1-3GalNAcαO(CH2)2CH2NH2

Polyacrylamide (PAA) conjugated, human serum albumin (HAS)-conjugated oramine conjugated glycoconjugates were utilized for glycan probepreparation. Glycoconjugates were synthesized chemoenzymaticallyaccording to methods described in Yu, H. et al., 2007. Org Biomol Chem.5:2458-63, the contents of which are herein incorporated by reference intheir entirety. Sialoglycans are synthesized using the “one-potthree-enzyme” approach as described by Yu et al (Yu, H. et al., NatProtoc. 2006. 1(5): 2485-92, Yu, H. et al., J Am Chem Soc. 2005.127:17618-9 and Yu, H. et al., 2006. Angew Chem Int Ed Engl. 45:3938-44,the contents of each of which are herein incorporated by reference intheir entirety). The compound structure was confirmed by HRMS (ESI) massspectrometry. Purity of each synthesized glycan was assessed by HPLCanalysis and only glycan preparations with greater than 95% purity wereused.

Arrays were printed on epoxide-derivatized slides (Corning, New York)with NanoPrint LM-60 Microarrayer equipped with 946MP3 MicroarrayPrinting Pins (Arrayit Corporation, Sunnyvale, California) with 16sub-array blocks on each slide. Glycan probes were distributed into384-well source plates using four replicate wells per sample and 8 µLper well. Glycan probes were prepared at a concentration of 100 µM perglycan in print buffer (300 mM Phosphate buffer, pH 8.4). Additionally,the linker (O(CH2)2CH2NH2) alone and buffer alone (300 mM phosphatebuffer, pH 8.4) were printed on the array in four replicates. To monitorprinting quality, murine IgG and human IgG were also printed on eachslide (40 and 20 ng/uL in PBS containing 10% glycerol, JacksonImmunoResearch Laboratories, WestGrove, Pennsylvania). The arrays wereprinted with four 946MP3 pins (5 µm tip, 0.25 uL sample channel,approximately 100 µm spot diameter, Arrayit Corporation). Each block(sub-array) had 10 rows, 8 columns with spot to spot spacing of 275 µm.The humidity level in the arraying chamber was maintained at about 70%during printing. Printed slides were left on the arrayer deck overnight,allowing humidity to drop to ambient levels (40-45%). The slides werethen packed, vacuum sealed and stored at room temperature.

The glycan array was assayed using: Hu2G12-2B2,L0H2, Hu8C2-2D6,L1H1,Hu5G2-1B3,L1H2, Mu2G12-2B2, and Mu3F1.

Additionally, control antibodies and lectins were also tested on thearray to determine if the glycans in the array can be recognized byknown glycan binding agents. These included: (a) Anti-Gc antibody, whichbinds to Gc containing glycans, Neu5Gcα6GalNAcαO(CH2)2CH2NH2 (GcSTn)(Glycan ID No.2), Neu5Gcα6Galβ4GlcNAcβO(CH2)2CH2NH2 (Glycan ID No. 4),Neu5Gcα6Galβ4GlcβO(CH2)2CH2NH2 (Glycan ID No. 6),Neu5Gcα6GalβO(CH2)2CH2NH2 (Glycan ID No. 8) andNeu5Gcα3Galβ1-3GalNAcαO(CH2)2CH2NH2 (Glycan ID No. 13); (b)MAL-II(Maackia Amurensis Lectin II) which binds to glycans containing(2,3)-linked sialic acid such as Neu5Acα3Galβ1-3GalNAcαO(CH2)2CH2NH2(Glycan ID No. 12) and Neu5Gcα3Galβ1-3GalNAcαO(CH2)2CH2NH2 (Glycan IDNo. 13); (c) SNA (Sambucus Nigra Lectin) which preferentially binds toglycans containing (2, 6) sialic acid linked to a terminal galactosesuch as Neu5Acα6Galβ4GlcNAcβO(CH2)2CH2NH2 (Glycan ID No.3),Neu5Gcα6Galβ4GlcNAcβO(CH2)2CH2NH2(Glycan ID No.4),Neu5Acα6Galβ4GlcβO(CH2)2CH2NH2(Glycan ID No.5) andNeu5Gcα6Galβ4GlcβO(CH2)2CH2NH2(Glycan ID No. 6); and (d) Palivizuamab asan isotype negative control and bound no glycans nor linker/bufferprinted controls as expected.

An epoxy blocking buffer (300 ml) was prepared by combining 15 ml of 2 MTris buffer (pH 8) with 0.9 ml of 16.6 M ethanolamine and 284.1 ml ofdistilled water. The solution was filtered using a 0.2 µM nitrocellulosemembrane. The epoxy buffer solution as well as 1 L of distilled waterwere pre-warmed to 50° C. Glass slides were arranged in a slide holderand quickly submerged in a staining tub with the warmed epoxy blockingbuffer. Slides were incubated in the epoxy blocking buffer for 1 hour at50° C. with periodic shaking to deactivate epoxy binding sites. Next,slides were rinsed with distilled water, placed into ProPlate slideholders (Grace Bio-Labs # 204862 16 square 7x7 mm chambers) and thenblocked with PBS with 1% OVA at 25° C. for one hour. Test antibodies andisotype control antibodies were tested at 1 and 2.5 ug/mL. Controlantibody anti-Gc was tested at 0.5 ug/mL and 1 ug/mL. Control biotintagged lectins were tested at 40 ug/mL for MALII and 20 ug/mL for SNA.All antibodies/lectins were diluted in blocking buffer (1% OVA/PBS) andincubated with the glycan array for one hour at 25° C. After extensivewashing, binding of polyclonal serum antibodies was detected byincubating glycan microarray slides with Cy3-conjugated anti-SA,anti-mouse IgG or antihuman IgG (Jackson Immunoresearch, West Grove, PA)for one hour. Slides were then washed extensively, dried and scannedwith a Genepix 4000B scanner (Laser at 100%; gain at 350; 10 µm pixels).Raw fluorescence intensity data from scanned images were extracted usingthe Genepix software and analysis of raw data was carried out.Antibodies were considered to be highly specific for AcSTn and GcSTn ifthey demonstrated binding to both molecules, but not to Tn or any otherglycans on the array.

Antibodies Hu2G12-2B2,LOH2, Hu8C2-2D6,L1H1, Hu5G2-1B3,L1H2, Mu2G12-2B2,and Mu3F1 demonstrated binding to AcSTn and GcSTn (Glycan ID No 1 and 2)but not to other glycans in the array demonstrating that theseantibodies have an affinity specifically for STn glycans only. Asexpected, the anti-Gc antibody, bound to all glycans in the arraycontaining Gc, MALII bound to glycans in the array containing(2,3)-linked sialic acid, SNA bound to glycans in the array containing(2, 6) sialic acid linked to a terminal galactose and the Palivizuamabcontrol showed no binding to the glycans. These results demonstratedthat the glycan array contains glycans that can be recognized byantibodies and proteins that are specific to the printed glycans.

Example 30: Neoglycolipid Array Analysis

Neoglycolipid probes are prepared from chemically synthesized glycansdescribed in table below.

TABLE 30 List of glycans Glycan ID No Glycan 1Neu5Acα6GalNAcαO(CH2)2CH2NH2 (AcSTn) 2 Neu5Gcα6GalNAcαO(CH2)2CH2NH2(GcSTn) 3 Neu5Acα6Galβ4GlcNAcβO(CH2)2CH2NH2 4Neu5Gcα6Galβ4GlcNAcβO(CH2)2CH2NH2 5 Νeu5Acα6Galβ4GlcβO(CΗ2)2CΗ2ΝΗ2 6Νeu5Gcα6Galβ4GlcβO(CΗ2)2CΗ2ΝΗ2 7 Neu5Acα6GalβO(CH2)2CH2NH2 8Νeu5Gcα6GalβO(CΗ2)2CΗ2ΝΗ2 9 GalNAcαO(CH2)2CH2NH2 10Galβ3GalNAcβO(CH2)2CH2NH2 11 Gal3βGalNAcαO(CH2)2CH2NH2 12Neu5Acα3Galβ1-3GalNAcαO(CH2)2CH2NH2 13Neu5Gcα3Galβ1-3GalNAcαO(CH2)2CH2NH2

Neoglycolipids are prepared by conjugating the glycans to aminophospholipid N-aminoacetyl-N-(9-anthracenylmethyl)-1,2-dihexadecyl-sn-glycero-3-phosphoethanolamine (ADHP) togenerate fluoresecent probes or withL-1,2-dihexadecyl-sn-glycero-3-phosphoethanolamine (DHPE) by reductiveamination orN-aminooxyacetyl-1,2-dihexadecyl-sn-glycero-3-phosphoethanolamine (AOPE)by oxime ligation to generate non fluoresecent probes. The conjugationreaction allows the NGLs to be immobilized on solid matrices. Glycansthat have been reductively released from glycoproteins are subject tomild periodate reaction prior to conjugation. After conjugation, the NGLproducts are purified to remove excess lipids and salts, analyzed bymass spectrophotometry and quantified on High Performance thin layerchromatography by densitometry.

Neoglycolipid arrays are produced by robitically dispensing theneoglycolipid probes onto nitrocellulose-coated glass slides in aliposome formulation. Probes are printed at multiple concentrations anddensities to determine the optimal hybridization conditions.

Slides are probed with purified anti-STn antibody solutions orpolyclonal serum containing anti-STn antibodies. Antibody binding isdetected using biotinylated secondary antibody followed by afluorescently labeled streptavidin. Slides are scanned using aProScanArray (Perkin Elmer Life Sciences), and Fluorescent bindingsignals are quantified using ScanArray Express software (PerkinElmerLife Sciences). Purified antibodies or sera are considered to be highlyspecific for AcSTn and GcSTn if they demonstrate binding to bothmolecules, but not to Tn or any other glycans on the array.

What is claimed is:
 1. An antibody, wherein said antibody comprises aheavy chain variable domain (VH) with a CDR-H3 complementaritydetermining region having at least 50% amino acid sequence identity toan amino sequence selected from the group consisting of SEQ ID NOs: 115,114, 116-120, 140, and
 141. 2. The antibody of claim 1, wherein the VHcomprises: a CDR-H1 having at least 50% sequence identity to an aminoacid sequence selected from the group consisting of SEQ ID NOs: 105,106, and 136; and a CDR-H2 having at least 60% sequence identity to anamino acid sequence selected from the group consisting of SEQ ID NOs:107-113, and 137-139.
 3. An antibody, wherein said antibody comprises alight chain variable domain (VL) with a CDR-L3 having at least 50% aminoacid sequence identity to an amino sequence selected from the groupconsisting of SEQ ID NOs: 89, 91, 93, 95-98, 101-103, 133-135, and 148.4. The antibody of claim 3, wherein the VL comprises: a CDR-L1 having atleast 50% sequence identity to an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 121-129, and 142-146; and a CDR-L2having at least 50% sequence identity to an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 77, 79-81, 83-86, 88, 130-132,and
 147. 5. The antibody of any of claims 1-4 comprising at least onehuman framework region having an amino acid sequence with at least 70%sequence identity to an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 206-216.
 6. An antibody comprising a VH havingat least 70% sequence identity to an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 220-224, 230-234, 237-241, 249-253,and 256-260.
 7. An antibody comprising a VL having at least 70% sequenceidentity to an amino acid sequence selected from the group consisting ofSEQ ID NOs: 217-219, 225-229, 235, 236, 242-248, 254, and
 255. 8. Theantibody of claim 6 or 7, wherein said antibody comprises: a VH havingat least 95% sequence identity to an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 220-224; and a VL having at least95% sequence identity to an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 217-219.
 9. The antibody of claim 6 or 7,wherein said antibody comprises: a VH having at least 95% sequenceidentity to an amino acid sequence selected from the group consisting ofSEQ ID NOs: 237-241; and a VL having at least 95% sequence identity toan amino acid sequence selected from the group consisting of SEQ ID NOs:235 and
 236. 10. The antibody of any of claims 1-9, wherein saidantibody comprises an isotype selected from the group consisting IgG1,IgG2a, IgG2b, IgG2c, IgG3, and IgG4.
 11. The antibody of any of claims1-10, wherein said antibody is a human or humanized antibody.
 12. Theantibody of any of claims 1-10, wherein said antibody is a human IgG1antibody.
 13. A construct encoding the antibody of any of claims 1-12.14. A cell comprising the construct of claim
 13. 15. A vector comprisingthe construct of claim
 13. 16. An antibody produced by the cell of claim14.
 17. The antibody of any of claims 1-12, wherein said antibody bindsto cell-associated STn with a half maximal effective concentration(EC50) of from about 0.01 nM to about 30 nM.
 18. The antibody of any ofclaims 1-12, wherein said antibody is conjugated to a therapeutic agent.19. The antibody of claim 18, wherein said therapeutic agent is acytotoxic agent.
 20. The antibody of claim 19, wherein said cytotoxicagent is selected from the group consisting of monomethyl auristatin E(MMAE) and monomethyl auristatin F (MMAF).
 21. The antibody of claim 20,wherein the cytotoxic agent is MMAE and wherein said antibody is capableof killing an STn-associated cell with a half-maximal inhibitoryconcentration (IC50) of from about 0.1 nM to about 20 nM.
 22. A methodof treating cancer comprising administering the antibody of any ofclaims 1-12 or 17-21.
 23. The method of claim 22, wherein said cancercomprises at least one tumor.
 24. The method of claim 23, wherein thevolume of said at least one tumor is reduced.
 25. The method of claim24, wherein the volume of said at least one tumor is reduced by at least20%.
 26. The method of any of claims 23-25, wherein said at least onetumor comprises at least one tumor cell, wherein said at least one tumorcell comprises at least one tumor-associated carbohydrate antigen(TACA).
 27. The method of claim 26, wherein said at least one TACAcomprises sialyl(α2,6)N-acetylgalactosamine (STn).
 28. The method of anyof claims 22-27, wherein said cancer comprises one or more of breastcancer, colon cancer, pancreatic cancer, lung cancer, cervical cancer,ovarian cancer, stomach cancer, prostate cancer, and liver cancer. 29.The method of any of claims 22-28, wherein the antibody is administeredin combination with a chemotherapeutic agent and/or therapeuticantibody.
 30. The method of claim 29, wherein the chemotherapeutic agentis selected from at least one of fluoropyrimidine, oxaliplatin, andirinotecan.
 31. The method of claim 28 or 29, wherein the therapeuticantibody is selected from at least one of bevacizumab and anti-epidermalgrowth factor receptor (EGFR) antibody.
 32. The method of any of claims22-31, wherein the antibody is administered at a dose of from about 0.1mg/kg to about 30 mg/kg.
 33. The method of claim 32, wherein theantibody is administered at a dose of from about 2.5 mg/kg to about 5mg/kg.
 34. The method of claim 32 or 33, wherein the antibody isdetectable in at least one subject sample obtained from about 1 dayafter treatment to about 1 month after treatment.
 35. The method ofclaim 34, wherein the antibody is conjugated with MMAE, and wherein thedrug to antibody ratio (DAR) of said MMAE to said antibody changes byless than 50% in said at least one subject sample.
 36. A method ofscreening a cell or sample for the presence of at least one TACA, saidmethod comprising contacting the cell or sample with the antibody of anyof claims 1-12.
 37. The method of claim 36, wherein said at least oneTACA comprises STn.
 38. The method of claim 36 or 37, wherein saidsample is a biological sample, said biological sample obtained from asubject.
 39. The method of claim 38, wherein said subject has or issuspected of having cancer.
 40. The method of claim 38 or 39, whereinsaid biological sample comprises one or more of a cell, a tissue, atissue section, and a body fluid.
 41. The method of any of claims 36-40,wherein said antibody comprises a detectable label.
 42. The method ofany of claim 36-40, wherein said antibody is detected using a detectionagent.
 43. The method of claim 42, wherein said detection agent is asecondary antibody.
 44. The method of claim 43, wherein said secondaryantibody comprises a detectable label.
 45. A method of diagnosing cancerin a subject comprising screening a sample according to the method ofany of claims 38-44.
 46. The method of claim 45, wherein said method ispart of a companion diagnostic.
 47. The method of claim 46, wherein saidcompanion diagnostic is used for one or more of stratifying cancerseverity, stratifying cancer risk, selecting a subject for a clinicaltrial, developing a therapeutic regimen, modulating a therapeuticregimen, increasing treatment safety, and modulating treatmenteffectiveness.
 48. The method of any of claims 36-40, wherein saidsample comprises a protein array.
 49. The method of claim 48, whereinsaid protein array comprises one or more antibodies configured to bindone or more proteins, wherein at least one of said one or more proteinspresent in said sample.
 50. A kit for carrying out the method of any ofclaims 36-49, said kit comprising the antibody of any of claims 1-12.51. The kit of claim 50 comprising a secondary antibody.
 52. The kit ofclaim 51, wherein said secondary antibody comprises a detectable label.53. A composition comprising one or more of the antibody of any ofclaims 1-12 and 17-21.
 54. The composition of claim 53 comprising atleast one excipient.
 55. The composition of claim 54, wherein said atleast one excipient comprises a pharmaceutically acceptable excipient.56. The composition of claim 53, wherein said composition comprises anantibody-coated agent.
 57. The composition of claim 56, wherein saidantibody-coated agent comprises one or more of a particle, ananoparticle, a protein, a fusion-protein, a lipid, a liposome, and acell.
 58. The composition of claim 56 or 57, wherein said antibody is anantibody fragment.
 59. The composition of claim 58, wherein saidantibody fragment is selected from one or more of an Fab fragment and asingle chain Fv.
 60. A modified cell comprising a synthetic construct,wherein said synthetic construct encodes a factor, wherein said factormodulates cellular STn levels.
 61. The modified cell of claim 60,wherein said factor comprises at least one factor involved in STnsynthesis.
 62. The modified cell of claim 61, wherein said at least onefactor is selected from at least one of(Alpha-N-Acetyl-Neuraminyl-2,3-Beta-Galactosyl-1,3)-N-Acetylgalactosaminide,Alpha-2,6-Sialyltransferase I (ST6GalNAc I), T-synthase, and Core 1Beta3-Galactosyltransferase-Specific Molecular Chaperone (COSMC). 63.The modified cell of any of claims 60-62, wherein said modified cellcomprises elevated STn levels when compared with at least one unmodifiedcell.
 64. The modified cell of claim 60, wherein said factor reducesexpression of ST6GalNAC.
 65. The modified cell of claim 64, wherein saidfactor is an inhibitory ribonucleic acid (RNA) molecule.
 66. Themodified cell of any of claims 60-65, wherein said modified cell is amodified ovarian tumor cell.
 67. The modified cell of claim 66, whereinsaid modified ovarian tumor cells is selected from one or more of aSKOV3 cell, an OVCAR3 cell, an OVCAR4 cell, a BRCA1 mutant tumor cell,and a non-BRCA1 mutant tumor cell.
 68. A method of characterizingbinding of an antibody, said method comprising contacting a glycan arraywith said antibody, wherein said glycan array includes a plurality ofglycans.
 69. The method of claim 68, wherein said glycan array comprisesa panel of glycans, wherein said panel of glycans consists of one ormore of each of: Neu5Acα6GalNAcαO(CH2)2CH2NH2;Neu5Gcα6GalNAcαO(CH2)2CH2NH2; Neu5Acα6Galβ4GlcNAcβO(CH2)2CH2NH2;Neu5Gcα6Galβ4GlcNAcβO(CH2)2CH2NH2; Neu5Acα6Galβ4GlcβO(CH2)2CH2NH2;Neu5Gcα6Galβ4GlcβO(CH2)2CH2NH2; Neu5Acα6GalβO(CH2)2CH2NH2;Neu5Gcα6GalβO(CH2)2CH2NH2; GaINAcaO(CH2)2CH2NH2;Galβ3GalNAcβO(CH2)2CH2NH2; Gal3βGalNAcαO(CH2)2CH2NH2;Neu5Acα3Galβ1-3GalNAcαO(CH2)2CH2NH2; andNeu5Gcα3Galβ1-3GalNAcαO(CH2)2CH2NH2.
 70. The method of claims 68 or 69,wherein each of said plurality of glycans are part of a neoglycolipidprobe.